Pregnant Mare Serum Gonadotropin Research Articles

Effect of Pregnant Mare Serum Gonadotropin and Antidopamine on the gonads of female bileh fish (Rasbora maninjau)

Bileh fish (Rasbora maninjau) still have difficulty spawning in a controlled manner in aquaculture containers because the maturation of female fish gonads is slow and takes a long time. Internal factors, including the hormonal system, strongly influence the maturation of fish gonads. This study aims to determine the effect of Pregnant Mare Serum Gonadotropin (PMSG) and antidopamine on female gonads of bileh fish. The research used an experiment with a CRD (Completely Randomized Design) experimental design. The experiment consisted of four treatments and three repetitions. The treatments were the administration of PMSG and antidopamine hormones at different doses through feed, namely: 0 ml/kg feed (P1), 0.5 ml/kg feed (P2), 1 ml/kg feed (P3) and 2 ml/kg feed (P4). The research procedures included container and fish preparation, hormone, and feed preparation, fish rearing, and data collection. The test parameters evaluated were weight gain (Wg), gonadosomatic index (GSI), and gonadal maturity level (GML). Data on test parameters were analyzed with statistics (Anova). The research results showed that the administration of PMSG hormones and antidopamine through feed had a significant effect on weight gain, gonadosomatic index and gonad maturity level of bileh fish (P<0.05). Effective and best treatment at P3 (1 ml/kg feed) with Wg 0.22grams, GSI 8.58%, and GML IV 60%.

Detrimental effects of electromagnetic radiation emitted from cell phone on embryo morphokinetics and blastocyst viability in mice.

Electromagnetic radiation (EMR) has deleterious effects on sperm motility and viability, as well as oocyte membrane and organelle structure. The aim was to assess the effects of cell phone radiation on preimplantation embryo morphokinetics and blastocyst viability in mice. For superovulation, 20 female mice were treated with intraperitoneal (IP) injections of 10 IU pregnant mare's serum gonadotropin (Folligon® PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) after 48 h. The zygotes (n = 150) from the control group were incubated for 4 days. The experimental zygotes (n = 150) were exposed to a cell phone emitting EMR with a frequency range 900-1800 MHz for 30 min on day 1. Then, all embryos were cultured in the time-lapse system and annotated based on time points from the 2-cell stage (t2) to hatched blastocyst (tHDyz), as well as abnormal cleavage patterns. Blastocyst viability was assessed using Hoechst and propidium iodide staining. Significant increases (P < 0.05) were observed in the cleavage division time points of t2, t8, t10, and t12 of the experimental group compared with the controls. In terms of blastocyst formation parameters, a delay in embryo development was observed in the experimental group compared with the controls. Data analysis of the time intervals between the two groups showed a significant difference in the s3 time interval (P < 0.05). Also, the rates of fragmentation, reverse cleavage, vacuole formation, and embryo arrest were significantly higher in the experimental group (P < 0.05). Furthermore, the cell survival rate in the experimental group was lower than the control group (P < 0.05). Exposure to EMR has detrimental consequences for preimplantation embryo development in mice. These effects can manifest as defects in the cleavage stage and impaired blastocyst formation, leading to lower cell viability.

Influence of repeated estrus synchronization treatment on hormone secretion, growth, and development of dairy goats.

In large-scale intensive farms, dairy goats often undergo frequent estrus synchronization (ES) treatment, which may result in a decline in reproductive performance; however, the underlying mechanism remains unclear. The present study aimed to investigate the effect of pregnant mare serum gonadotropin (PMSG) and progesterone (P4)-mediated ES treatment on fertility in dairy goats, while also identifying key metabolic and endocrine mechanisms that influence reproductive performance in does subjected to repeated ES treatment. Forty-eight Saanen does were randomly assigned to two groups (24 goats each) that received ES treatments either thrice fortnightly (3-PMSG) or once (1-PMSG) simultaneously with the third ES treatment of the 3-PMSG group during the breeding season. ES treatment was performed via the intravaginal insertion of a controlled internal drug release (CIDR) device impregnated with 300 mg P4, followed by 300 IU PMSG injections 48 h before CIDR withdrawal. Blood was collected to detect the level of hormones and blood biochemical indices. Additionally, estrus rate, fecundity rate, body weight, size, and lactation performance were measured. The results showed that repeated ES treatment markedly decreased the estrus rate and fecundity rate of goats. Among the does in all groups, there was no substantial difference in follicle stimulating hormone, luteinising hormone, gonadotropin-releasing hormone, melatonin, growth hormone, PMSG, total cholesterol, total protein, and glucose levels, as well as the body weight, body size, and lactation performance. Repeated ES treatment elevated estrogen (E2) levels 36, 48, and 72 h post-CIDR removal; increased P4 upon CIDR insertion; and raised PMSG antibody levels 24, 48, and 72 h post-CIDR removal. The results suggest that elevated anti-PMSG levels are the primary reason for the decline in ES efficiency, and that high E2 and P4 levels at some time points also impair reproductive performance. These findings provide novel insights into the metabolic effects of repeated PMSG stimulation in goats, guiding future reproductive hormone use in breeding practices.

Effect of ovarian stimulation on developmental speed of preimplantation embryo in a mouse model.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the Stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

Effects of acupuncture on RT-BM1 and maternal-fetal immune tolerance in rats with ovarian hyperstimulation: 针刺对超排卵大鼠RT-BM1及母胎免疫耐受的影响

ObjectiveThis study aimed to evaluate the effects of acupuncture on embryo implantation and maternal–fetal immune tolerance in rats with ovarian hyperstimulation, identify the mechanisms of acupuncture treatment, and promote application of acupuncture in the field of assisted reproduction. MethodsEighty female Wistar rats were randomized into normal (N), model (M), acupuncture treatment (A), and progesterone treatment (P) groups, with 20 rats in each group. An ovarian hyperstimulation model was established in groups M, A, and P by the peritoneal injection of pregnant mare serum gonadotropin (PMSG) combined with human chorionic gonadotropin (HCG). Bilateral “Sanyinjiao (SP6)”, “Zusanli (ST36)” and “Taichong (LR3)” as acupoints were needled in rats in group A. Rats in group P received an intramuscular injection of progesterone (4 mg/kg). All pregnant rats were treated for seven days. On the 8th day, the pregnant rats were sacrificed for testing. The expression levels of estrogen (E2) and progesterone (P4) in serum and their receptors in the endometrium, Th1 and Th2 cytokines in serum and endometrium, RT-BM1 (the counterpart of HLA-G in rats) mRNA, Ly49 activating receptors (Ly49s3, Ly49s5) and inhibitory receptors (Ly49i3, Ly49i4, Ly49i5) on immunocytes in the maternal–fetal interface were detected using ELISA, Western blot, and real-time polymerase chain reaction (PCR). ResultsCompared with group N, the pregnancy rate and the number of implanted embryos were significantly decreased in group M (P < 0.05); E2 and P4 levels in serum, and the protein levels of ER-α and Progesterone Receptor (PGR) in the implantation site of endometrial tissue were significantly reduced in group M (P < 0.05); the expression of INF-γ and IL-2 in serum and endometrium were significantly increased, while the expression of IL-4, IL-6, and IL-10 in serum and endometrium were significantly decreased in group M(P < 0.05). The mRNA levels of RT-BM1, Ly49i3, and Ly49i4 receptors in group M were significantly higher than those in group N, whereas the mRNA levels of Ly49i5 and Ly49s5 receptors in group M were significantly lower than those in group N at the maternal–fetal interface (P < 0.05). The pregnancy rate and number of implanted embryos were significantly higher in group A than in group M (P < 0.05). Serum E2 and P4 levels were significantly higher in groups A and P than in group M (P < 0.05). The expression of ER-α and PGR in the implantation site of endometrial tissue in group A were significantly higher than those in group M (P < 0.05). The serum levels of INF-γ and IL-2 in group A and group P were significantly lower than those in group M (P < 0.05). The serum expression of IL-4, IL-6, IL-10 in group A and group P were significantly higher than those in group M (P < 0.05). The endometrium expression of INF-γ and IL-2 in group A were significantly lower than those in group M (P < 0.05). The endometrium expression of IL-4, IL-6, IL-10 in group A were significantly higher than those in group M (P < 0.05). The endometrium expression of INF-γ in group P were significantly lower than those in group M (P < 0.05). The endometrium expression of IL-6 and IL-10 in group P were significantly higher than those in group M (P < 0.05). The mRNA levels of RT-BM1 and these Ly49 receptors (except the Ly49s3 receptor) at the maternal–fetal interface of groups A and P were higher than those of group M (P < 0.05). ConclusionAcupuncture can significantly increase the pregnancy rate and average number of implanted blastocysts in rats with ovarian hyperstimulation. This mechanism is related to the effects of acupuncture in upregulating the expression of RT-BM1, regulating the balance of Th1/Th2 cytokines, and upregulating the expression of inhibitory receptors in immunocytes.

#236 : Alleviating Effects of EGCG on Ovarian Hyperstimulation Syndrome and the Potential Modulating Mechanism

Background and Aims: Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications of COH during IVF treatment. Increased capillary permeability is a hallmark of the pathophysiology of OHSS, vascular endothelial growth factor (VEGF) is an important mediator in OHSS, and studies have established a correlation between serum VEGF levels and the severity of the OHSS. (-)-epigallocatechin-3-gallate (EGCG), the most prevalent and biologically active polyphenolic catechin in green tea, has been reported to produce a number of beneficial effects on humans. Numerous studies have shown that in many pathological processes, EGCG could inhibit VEGF and its receptor expression and have an angiogenesis effect. Herein, EGCG might have a therapeutic effect on OHSS. Methods: We investigated the role of EGCG in OHSS both in vitro and in vivo. Primary human granulosa-lutein (hGL) cells and a human granulosa-like tumor (KGN) cell line were cultured and treated for 24 hours with various concentrations of EGCG. In SD rats, an animal OHSS model was established by injecting pregnant mare serum gonadotropin (PMSG) and randomly assigning them to receive vehicle only or EGCG for three days. For comparison, all experiments were repeated 3-8 times. The effect of EGCG on KGN and hGL cells was determined by MTT assay. The body weight of rats was measured every day, and the ovary size and weight were measured after removal. Evans-blue was used to determine permeability. ELISA was used to measure serum estrogen and VEGF levels in rats. RNA and protein expressions of VEGF, VEGFR-2, TGF-[Formula: see text]1, and T[Formula: see text]R II were detected by qPCR and Western-blotting and immunostaining in vitro and in vivo experiments. Results: Our research found that giving rats EGCG prevented the development of OHSS, as evidenced by histological examination, ovarian weight, and morphology. When compared to the OHSS group, the EGCG treatment group had significantly lower ovary weight (147.9 vs 206.5 mg) (Fig. 1). Furthermore, when compared to OHSS rats, EGCG-treated rats have lower ovarian VEGF expression as measured by IHC and RT-qPCR. VEGF and E2 protein levels in the serum of the EGCG treatment group are significantly lower. EGCG exerted inhibitory effects on cell growth only in high dose (50 uM) and longtime (48 h) treatment in KGN and hGL cells. In KGN cells and hGL cells, EGCG significantly reduces the expression of VEGF and TGF-[Formula: see text] at the RNA and protein levels. Furthermore, EGCG inhibits TGF-[Formula: see text]1-induced VEGF production and secretion in KGN cells by suppressing TGF-[Formula: see text] expression and its traditional Smad signaling pathway. EGCG also downregulates VEGF expression through the 67-kDa laminin receptor-mediated PKA-CREB pathway (Fig. 2). Conclusions: EGCG inhibited VEGF via the TGF-[Formula: see text]1 classical-SMAD pathway and the 67LR-mediated CREB pathway, reducing ovarian inflammation and slowing the progression of OHSS in a rat model.

Maturation of Female Yellow Rasbora (&lt;i&gt;Rasbora Lateristriata&lt;/i&gt; Bleeker, 1854) Using Oodev at Different Doses in Feed

The current high demand for Yellow rasbora (Rasbora lateristriata) is not supported by the availability of captured Yellow rasbora in nature. Aquaculture is the most rational way of utilizing biological natural resources. In intensive aquaculture, it is necessary to optimize all processes that occur in aquaculture, including hatchery. However, the common problem that often happens in hatchery activities is spawning which depends on the season. The hormonal manipulation technique is an appropriate way to stimulate gonadal maturation. Oodev is a hormonal combination of pregnant mare serum gonadotropin and anti-dopamine to stimulate gonadal maturation. The purpose of this study was to determine the effectiveness of using the Oodev with different doses in feed to accelerate gonad maturation of female Yellow rasbora. The study was carried out with four treatments and three replications in 21 days with different doses of Oodev, such as; A (Feed without Oodev), B (0.5 mL/kg feed), C (1 mL/kg feed) and D (2 mL/kg feed). The parameters observed in this study were gonad maturity level, histological structure of ovary, gonadosomatic index, fecundity, and diameter of eggs. The results showed that the dose of Oodev at 1.0 mL/kg feed was an effective dose to optimize the gonad maturity of female Yellow rasbora. This is proven by the highest results shown on all parameters, such as; the maturity level in the IV phase, histological structure of the ovary which showed the dominance of the oocyte maturation phase, gonadosomatic index of 14.014%, the fecundity of 721 eggs, and egg diameter of 0.865 mm. In conclusion, using Oodev in feed at a dose of 1.0 ml/kg of feed for 21 days is an effective dose to optimize the maturation of female Yellow rasbora.

Effect of carbofuran insecticide administration to fetal weight and length of mice (Mus musculus)

Introduction: Carbofuran could decrease cholinesterase levels are significantly associated with reduced gestation duration of and the risk of low birth weight and length. This study aims to determine a decrease in the fetal weight and length of pregnant mice exposed to carbofuran. Objective: This study used 18 female mice aged 10 weeks old with a weight of 25-30 grams. Female mice were mated with 18 male mice aged 12 weeks old which were synchronized using the hormones of Pregnant Mare Serum Gonadotrophin (PMSG) and Human Chorionic Gonadotrophin (HCG). This study was divided into three treatments, namely control (C), carbofuran at a dose of 0.0417 mg/mouse (T1), and carbofuran at a dose of 0.0208 mg/mouse (T2). Each pregnant mouse was treated with carbofuran for 10 days from day 6 up to day 15 of gestation when vaginal plugs were seen. Fetal collection was carried out on day 17 to measure the fetal weight and length. Data were analyzed using the ANOVA test and Duncan's test (significance 5%). Results: The results showed that the treatment groups (T1 and T2) had a decrease in the fetal weight and length compared to the control (C) (p&lt;0.05), nevertheless the treatment groups (T1 and T2) did not have significant difference (p&gt;0/05). Conclusion: It can be concluded that administration of carbofuran to pregnant mice could decrease the fetal weight and length.

Acupuncture increased the Number of Retrieved Oocytes in a Mouse Model of POR: The Involvement of DNA Methylation in the Oocytes

Background:: Poor ovarian response (POR) reduces the success rate of in vitro fertilization mainly because of fewer oocytes retrieved. Acupuncture (Ac) therapy can improve the number of retrieved oocytes in the controlled ovarian stimulation program. The role of Ac in the corresponding epigenetic mechanism of POR has not been studied. Objective:: This study was conducted to determine the effect of Ac on the number of retrieved oocytes and its role in DNA methylation in a mouse model of POR. Methods:: Forty C57BL/6N female mice with normal estrous cycles were randomly classified into 4 groups of 10 each: control (Con) group, Ac-Con group, POR group, and Ac-POR group. Mice in POR and Ac-POR groups received a gastric gavage of Tripterygium wilfordii polyglycoside suspension of 50 mg/kg-1 once a day for 14 consecutive days. Ac was applied at “Shenting” (DU 24), “Guanyuan” (CV 4), “Zusanli” (ST 36), and “Shenshu” (BL 23) in the Ac-POR group for 10 min per session, once a day for 14 consecutive days. All four groups were stimulated with pregnant mare serum gonadotropin and human chorionic gonadotropin, and the number of retrieved oocytes and proportion of mature oocytes were recorded. The DNA methylation level in a single mouse oocyte in each group was analyzed using single-cell genome-wide bisulfite sequencing (scBSseq), and key pathways were identified using GO and KEGG enrichment analyses. Results:: A dissecting microscope revealed that the Ac therapy improved the number of retrieved oocytes compared with the POR group (p &lt; 0.05). ScBS-seq showed that there was no significant change in global DNA methylation levels between the POR model and control group mice. However, differences were primarily observed in the differentially methylated regions (DMRs) of each chromosome, and Ac decreased global DNA methylation. DMR analysis identified 13 genes that may be associated with Ac treatment. Cdk5rap2 and Igf1r, which mediate germ cell apoptosis, growth, and development, maybe most closely related to the Ac treatment of POR. KEGG analysis revealed that differentially expressed genes were mainly enriched in Wnt, GnRH, and calcium signaling pathways. The genes were closely related to the regulation of POR via Ac. Conclusion:: The results suggest that DNA methylation in oocytes is related to the development of POR and that the role of Ac in affecting DNA methylation in oocytes is associated with the Wnt, GnRH, and calcium signaling pathways as well as Cdk5rap2 and Igf1r in POR mice.

SAT415 Development Of A Novel, Preclinical Swine Model Of Transgender Men To Assess Testosterone Impact On Insulin Sensitivity And Whole-Body Metabolism

Abstract Disclosure: N. Mahabir: None. I. Ahmad: None. T. Wright: None. G. Ten Have: None. N. Deutz: None. A.E. Newell-Fugate: None. The effects oftestosterone therapy on peripheral insulin sensitivity in transgender men areequivocal due to heterogeneity of the population, short term patientfollow-up, and small cohort sizes. Most research has found testosterone therapyto decrease or have no effect on insulin sensitivity, but in one studytransgender men had increased insulin sensitivity. We have found thatshort-term, virilizing doses of testosterone in female pigs suppress circulatinginsulin levels. The objective of the current study was to develop a long-term,preclinical model of transgender men to assess insulin sensitivity and whole-bodymetabolism. A swine model of transgender men is superior to other models,because pigs closely mimic human cardiometabolic physiology and their largebody size permits longitudinal sampling and interventional procedures. Togenerate stable, long-term serum testosterone (TEST) concentrations in femalepigs, we fabricated testosterone silastic implants at the following doses:placebo (30 mm, n=1) or 15 mm (n=2), 30 mm (n=2), 60 mm (n=2), or 120 mm (n=1)TEST. Eight sexually mature female pigs were administered 14 days of progestin(Altrenogest) which was withdrawn on day 15. On day 16, 400 I.U. of pregnantmare serum gonadotropin and 200 I.U of human chorionic gonadotropin (PG600®,Merck Animal Health) were administered intramuscularly to stimulatefolliculogenesis and ovulation. Pigs had estrus detection performed dailythroughout the study and came into estrus 3-7 days post PG600® injection.Twenty-four hours post-estrus, implants were surgically placed subcutaneously,blood was taken, and biopsies of subcutaneous white adipose tissue werecollected from the incision site for baseline assessment of mitochondrialoxygen flux (Oroboros) and proteomic measurement of mitochondrial and insulinpathway abundance. After implant placement, fasted blood was collected at 24,48, 72 hours and every three days thereafter for six weeks. During weeks 2-6post-implantation, continuous glucose monitoring was performed (Freestyle Libre3). Twenty-four hours prior to study completion, a catheter was placed in thecranial vena cava and an intravenous glucose tolerance test was performed (0,5, 10, 20, 30, 40, 50, 60 min). The next morning fasted blood was collected (time0) and a stable isotopic tracer bolus was administered to measure whole bodyproduction of glycerol, branched chain amino acids, branched chain keto acids, and β-hydroxyβ-methylbutyric acid. After tracer bolus, blood was collected at 10, 20, 40, 60and 120 minutes. At 120 minutes post-bolus, tissues were collected for isotopiccompound measurement, mitochondrial function assays, and insulin signalingpathway assessment. This translational swine model will help uncover themechanistic effects of testosterone therapy on insulin sensitivity and whole-bodymetabolism in female to male transitioning patients. Presentation: Saturday, June 17, 2023

Use of deslorelin acetate to advance ovulation in goats for timed artificial insemination

The objective of this study was to determine the efficacy of deslorelin in advancing ovulation for timed artificial insemination (AI) protocols in goats. We hypothesized that deslorelin treatment advances the onset of ovulation and improves AI pregnancy rates. Does were synchronized using a 5-day CIDR (controlled internal drug releasing insert) protocol with prostaglandin treatment at CIDR insertion. For Experiment 1, does received 0.2 mg of intramuscular deslorelin (n = 9) or saline (control, n = 10) at CIDR removal. Serial blood collections and transrectal ultrasonography were performed to assess ovarian dynamics and identify ovulation. For Experiment 2, does received 0.2 mg of intramuscular deslorelin (n = 42), 5 ml of PMSG (pregnant mare serum gonadotropin)/hCG (human chorionic gonadotropin) (n = 42), or were left untreated (control, n = 42) at CIDR removal, and were subsequently bred by transcervical AI with fresh semen after 48 to 56 hours. Pregnancy diagnosis was performed at 50 and 90 days after AI. In Experiment 1, compared to control does, deslorelin-treated does had an increased (p &lt; 0.01) number of ovulations and increased (p &lt; 0.01) serum estradiol concentrations from 48 to 72 hours after CIDR removal. Serum progesterone concentrations did not differ between treatments. In Experiment 2, there was a main effect (p = 0.02) of treatment on pregnancy rates, with control does tending (p = 0.06) to have greater pregnancy rates than those treated with deslorelin or PMSG/hCG. Deslorelin treatment also resulted in decreased (p = 0.04) breeding season pregnancy rates and increased (p = 0.05) number of cycles to achieve pregnancy compared to control does. These results demonstrated that deslorelin not only has a super-ovulatory effect but also, at the dose given, can negatively impact does’ subsequent ovarian function and ability to achieve pregnancy.

FOXG1 is involved in mouse ovarian functions and embryogenesis

TGF-β superfamily has long been demonstrated to be essential for folliculogenesis and luteinization. Forkhead box G1 (FOXG1, also known as BF1), a member of the FOX family and an inhibitor of TGF-β signaling pathway, is a nucleocytoplasmic transcription factor that is essential for forebrain development. FOXG1 is involved in neurodevelopment and cancer pathology, however, little is known about the role of FOXG1 in reproduction. In this study, the spatiotemporal expression pattern of FOXG1 was examined during early mouse oocyte and embryonic development and its role during corpora luteum (CL) formation was further elucidated. The results showed that FOXG1 is localized in oocytes, theca cells (TCs) and CLs. After fertilization, FOXG1 is expressed at all stages during early embryogenesis, from zygotes to blastocysts. Following gonadotropin administration in immature mice, the expression of Foxg1 significantly increased along with steroidogenic genes, including Star, Hsd3β, Cyp11a1, as well as Cyp17a1 and Cyp19a1. The latter two first increased after pregnant mare serum gonadotropin stimulation, then decreased in response to hCG treatment. In addition, silencing of Foxg1 significantly reduced the concentration of testosterone and estrogen in cultured primary granulosa cells (GCs) and TCs (P < 0.05). Mechanistic studies demonstrated that the expression level of genes that are critical in estrogen synthesis were significantly reduced after Foxg1 silencing, including Cyp17a1 and Cyp19a1. In conclusion, FOXG1 is expressed in a stage-specific manner during folliculogenesis and embryogenesis and exerts a regulatory influence on testosterone and estrogen synthesis.

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation.

Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs) provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim of this study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouse oocytes using human embryonic stem cells conditioned medium (HESCM). In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI female mice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocytes without cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also 120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II (MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cell embryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalized estimating equations (GEE) method that calculated their rate ratio. Our data indicated there are significant differences between the maturation rates in HESCM and HESM (P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00). Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVM outcomes in mice.

In view of ovarian steroidogenesis and luteal construction to explore the effects of Bushen Huoxue recipe in mice of ovarian hyperstimulation

Ethnopharmacological relevanceBushen Huoxue recipe (BSHXR) is a widely used prescription medicine for treating gynecological diseases. We have previously found that BSHXR can improve the pregnancy outcome of controlled ovarian hyperstimulation (COH) mice by modulating the abnormal high level of progesterone. While the pharmacological mechanism of such therapeutic effect is not clear. Aim of the studyWe aimed to investigate the effects of BSHXR on the ovarian steroidogenesis and luteal function in mice undergoing COH. Materials and methodsA COH mouse model was established via an intraperitoneal injection of 0.4 IU/g pregnant mare serum gonadotropin (PMSG) and 1 IU/g human chorionic gonadotropin (HCG). The histological features of ovaries were observed using hematoxylin-eosin staining. The expression levels of FSHR, LHCGR, and key molecules in ovarian steroidogenesis, including CYP11A1, CYP17A1, CYP19A1, HSD3B1, and StAR, were examined via immunohistochemical staining, western blotting, and RT-qPCR. CD31, VEGFA, and FGF2 levels were assessed to evaluate ovarian vascularization. The protein and mRNA levels of ovarian ERK1/2, p-ERK1/2, MEK1/2, and p-MEK1/2 were also detected using western blotting, RT-qPCR, or immunofluorescence staining. ResultsCOH mice had a significantly increased volume and weight of the ovary and number of corpora lutea. In particular, COH exhibited a long-term influence on ovarian FSHR and LHCGR expression, disrupting the levels of CYP11A1, HSD3B1, and CYP17A1, causing poorer luteal angiogenesis. Compared with normal mice, the expression levels of ovarian VEGFA and FGF2 in COH mice were considerably lower on Day 1 after PMSG. On concomitant HCG treatment, both VEGFA and FGF2 expression surged dramatically on ED1 and then declined on ED4 and ED8. Moreover, the expression pattern of MEK1/2-ERK1/2 was almost consistent with that of VEGFA and FGF2. After treatment, BSHXR increased ovarian LHCGR, FSHR, CYP11A1, HSD3B1, and CYP17A1 levels, boosted luteal vascularization, and restored MEK1/2-ERK1/2 signaling in COH mice. ConclusionBSHXR restored the abnormally high progesterone level by regulating the CYP11A1 and HSD3B1 expression as well as promoted luteal angiogenesis, which was related with LHCGR-MEK1/2-ERK1/2-VEGFA/FGF2 signaling pathway in the ovary. This effect prevented the fluctuation of sex hormones in COH mice and benefited the outcome of pregnancy.

Optimization and Validation of a Liquid Formulation for a New Recombinant Veterinary Product using QbD Approach

Protein formulation and drug characterization are one of the most difficult and time-consuming tasks because of the complexity of biotherapeutic proteins. Hence, maintaining a protein drug in its active state typically requires preventing changes in its physical and chemical properties. Quality by Design (QbD) is a systematic approach emphasizing product and process understanding. Design of Experiments (DoE) is one of the most important QbD tools, allowing the possibility to modify the formulation attributes within a defined design space. Here, we report the validation of a RP-HPLC assay for recombinant equine chorionic gonadotropin (reCG) that demonstrated a high correlation with the in vivo potency biological assay. QbD concepts were then applied to obtain an optimized liquid formulation of reCG with a predefined quality product profile. The developed strategy demonstrates the importance of applying multivariable strategies as DoE to simplify formulation stages, improving the quality of the obtained results. Moreover, it is important to highlight that this is the first time that a liquid formulation is reported for an eCG molecule, since, up to now, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG) presented as a lyophilized product.

P-631 Alleviating effects of EGCG on ovarian hyperstimulation syndrome and the potential modulating mechanism through the VEGF pathway

Abstract Study question What are the underlying mechanisms of EGCG to alleviate Ovary hyperstimulation syndrome (OHSS)? Summary answer EGCG inhibited VEGF via TGF-β1 classical-SMAD pathway and 67LR-mediated CREB pathway and could reduce the ovarian inflammatory effect and attenuated OHSS progress in rat model. What is known already Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications of COH during IVF treatment. The pathophysiology of OHSS is characterized by increased capillary permeability, VEGF is an important mediator in OHSS, and serum VEGF levels have been shown to correlate with OHSS severity. (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active polyphenolic catechin in green tea and has been reported to have multiple effects in humans. Many research indicated that in many pathological processes, EGCG could inhibit VEGF and its receptor expression and have an angiogenesis effect. Herein, EGCG might have a therapeutic effect on OHSS. Study design, size, duration We investigated the role of EGCG in OHSS in vitro and in vivo. The primary human granulosa-lutein(hGL) cells and human granulosa-like tumor (KGN) cell line were cultured and treated with different concentrations of EGCG for 24 hours. Animal OHSS model was established in SD rats by injection of pregnant mare serum gonadotropin (PMSG), and randomly assigned to receive vehicle only or EGCG for 3 days. All experiments were performed 3-8 times for comparisons. Participants/materials, setting, methods The effect of EGCG on KGN and hGL cells was determined by MTT assay. The body weight of rats was measured every day, and the ovary size was measured after removal, the permeability was determined by Evans-blue. The serum estrogen and VEGF level in rats were detected by ELISA. RNA and protein expressions of VEGF, VEGFR-2, TGF-β, and TβR II were detected by qPCR and Western-blotting and immunostaining in vitro and in vivo experiments. Main results and the role of chance Our study demonstrated that administration of EGCG attenuated the development of OHSS in rats, as shown by histological examination and ovarian weight and morphology. The ovary weight was significantly decreased in the EGCG treatment group compared with the OHSS group (147.9 vs 206.5 mg). Additionally, compared to OHSS rats, EGCG treated rats exhibit downregulated ovarian VEGF expression determined by IHC and RT-qPCR. VEGF and E2 protein levels significantly decrease in the serum of the EGCG treatment group. EGCG exerted inhibitory effects on cell growth only in high dose (50uM) and longtime (48h) treatment in KGN and hGL cells. In KGN cells and hGL cells, EGCG significantly reduces the expression of VEGF and TGF-β at the RNA and protein levels. Furthermore, EGCG inhibits TGF-β1-induced VEGF production and secretion in KGN cells by suppressing TGF-β expression and its traditional Smad signaling pathway. EGCG also downregulates VEGF expression through the 67-kDa laminin receptor-mediated PKA-CREB pathway. Limitations, reasons for caution EGCG has been reported to employ a wide range of biological effects. Therefore, its suppressive effects on VEGF and TGF-β signaling pathway might be part of the pharmacological mechanisms to alleviate Ovary hyperstimulation syndrome. Further studies are also needed to explore the therapeutic mechanisms of melatonin from other perspectives. Wider implications of the findings Our findings add mechanistic insight in support of using EGCG as an adjuvant therapy in the management of OHSS. EGCG shows the potential to act as a novel alternative therapeutic drug to treat OHSS. Daily intake of green tea might be beneficial for women under COH to prevent OHSS. Trial registration number Not applicable

Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

NUCB2/nesfatin-1 suppresses the acrosome reaction in sperm within the mouse epididymis

ABSTRACT Nesfatin-1, a polypeptide hormone derived from the nucleobindin 2 (NUCB2) precursor protein, is known to regulate appetite and energy metabolism. Recent studies have also shown that NUCB2/nesfatin-1 is expressed in the reproductive organs of mice. However, the expression and potential role of NUCB2/nesfatin-1 in the mouse epididymis remain unclear. Therefore, we investigated the expression of NUCB2/nesfatin-1 in the mouse epididymis and its potential function. NUCB2/nesfatin-1 was detected in the epididymis by qRT-PCR and western blotting, and high expression levels were observed in epididymal epithelial cells by immunohistochemical staining. Pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) injections significantly increased NUCB2/nesfatin-1 expression in the epididymis. After castration, NUCB2/nesfatin-1 expression in the epididymis decreased, but was significantly increased by testosterone injection. Nesfatin-1-binding sites were found in the middle piece of testicular sperm, but were scarcely detected in the sperm head. By contrast, nesfatin-1 binding sites were identified on the sperm head within the epididymis. Furthermore, nesfatin-1 treatment inhibited the acrosome reaction in epididymal sperm. These results suggest that the nesfatin-1 protein produced in the epididymis binds to nesfatin-1 binding sites on the sperm head and plays a role in suppressing the acrosome reaction before ejaculation.

Immunohistochemistry of Leukemia Inhibitory Factor and Integrin αVβ3 in Mouse Endometrium Following Kisspeptin-54 Ovulation Trigger.

Kisspeptin (KP) is a group of hypothalamic neuropeptides encoded by KISS-1 gene. KP-54, a 54-amino-acid peptide, helps regulate the hypothalamic-pituitary-ovarian axis and plays a potential role in implantation. C57BL/6J female mice were superovulated via intraperitoneal injection of 5 International Units (IU) pregnant mare serum gonadotrophin (day 1). Forty-eight hours later, mice (5/group) were injected with phosphate-buffered saline (PBS) (group A), 5IU human chorionic gonadotrophin (hCG) (group B), or 3nmol KP-54 (group C). On day 7, mice were euthanized and uteri excised to create paraformaldehyde-fixed paraffin-embedded sections that were immunostained for the implantation markers: leukemia inhibitory factor (LIF) and integrin αVβ3 (ITG αVβ3). Slides were scored for intensity of staining in endometrial glandular epithelium (GE) and stromal cells (SCs) via histoscore (H-score). Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise comparisons. LIF expression was significantly higher in GE and SCs of mice triggered with KP-54 compared to placebo (P = .009 for both), but only higher than hCG trigger group in SCs (P = .009). Meanwhile, ITG αVβ3 expression was significantly higher in SCs of mice triggered with KP-54 compared to placebo (P = .028). In conclusion, using KP-54 as an ovulation trigger resulted in higher expression of the implantation markers LIF and ITG αVβ3 in mice endometrium compared to hCG or placebo. This suggests a potential role for KP-54 trigger in improving embryo implantation in clinical IVF. However, further studies are needed to correlate these results with clinical implantation rates and pregnancy outcomes.

Bioactivity and Pharmacodynamics of X002, a Follicle-stimulating Hormone-IgG4 Fc Fusion Protein.

Current follicle-stimulating hormone (FSH) drugs meet safety criteria but have suboptimal efficacy, poor patient compliance, and high cost. Alternative FSH-like drugs would help to meet the high market demand. Here, we evaluated X002, an FSH-Fc fusion protein, for bioactivity and half-life in vitro and in vivo. In all cases, the effects of X002 were compared with those of a commercially available short-acting FSH recombinant hormone. First, female Kunming mice (age, 21 to 24 d) were stimulated with pregnant mare serum gonadotropin (PMSG) for 46 h, after which naked oocytes were harvested, treated with X002 or the comparison agent at 37 °C for 4 h, and then evaluated for germinal vesicle breakdown. Second, cumulus-oocyte complexes (COC) were collected from PMSG-stimulated mice and cocultured with X002 or the comparison agent for 14 h; the COC diameters were then measured, and the expression of genes involved in COC expansion were evaluated using quantitative RT-PCR analysis. Third, to assess the pharmacokinetics of X002, female Sprague-Dawley rats (age, 6 to 8 wk) were injected subcutaneously with X002 or the comparison agent; serum samples then were collected at various times and assessed via ELISA. Fourth, to evaluate X002 pharmacodynamics, 26-d-old female Sprague-Dawley rats were treated with X002 or the comparison agent; 84 h later, the rats were stimulated with human chorionic gonadotropin (hCG). At 12 h after hCG injection, euthanasia was performed. Ovaries were removed and weighed, and serum levels of estradiol and progesterone were measured. Finally, to assess superovulation, the oocytes in the fallopian tubes were counted at 108 h after in vivo treatment of rats with X002 or the comparison agent. The data show that X002, a long-acting agent, promoted germinal vesicle breakdown and COC expansion in vitro and in vivo ovarian weight gain and superovulation to a degree similar to the short-acting comparison agent.