Choice of protocol for the in vivo bioassay of equine Chorionic Gonadotropin (eCG / PMSG) in immature female rats

To determine the role of the thyroid gland on the ovarian functions during the initiation process of puberty, we examined the effects and its mechanisms of hypothyroidism on the first ovulation induced by equine chorionic gonadotropin (eCG) in immature female rats. Animals were thyroidectomized on day 22 and were injected with 5 IU of eCG on day 26 to induce the first ovulation on day 29. The number of antral follicles that secrete inhibin and the ovarian weight were significantly increased in thyroidectomized rats (Tx rats) 48 h after eCG treatment compared with those in non-Tx rats. However, thyroidectomy (Tx) significantly suppressed the rates of ovulating animals on day 29. The blockage of ovulation in Tx rats was recovered by administration of human chorionic gonadotropin or luteinizing hormone (LH)-releasing hormone (LHRH) on day 28. Inhibition of serum LH (not follicle-stimulating hormone) levels induced by Tx was almost restored to control levels by injection of LHRH. A significant increment in prolactin levels in Tx rats was also observed on day 28. The present data indicate that Tx before puberty in female rats causes the blockage of the first ovulation and that the inhibitory effects on ovulation are mainly due to the reduction in the preovulatory LH surge, which is partially mediated through an inhibition of LHRH action on the secretion of LH.

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Inhibition by Indomethacin of the Already Accelerated Secretion of Inhibin and Estradiol in Equine Chorionic Gonadotropin-Primed Immature Female Rats.

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.16.483 ◽

1993 ◽

Vol 16 (5) ◽

pp. 483-485 ◽

Cited By ~ 2

Author(s):

Hiroshi KOGO ◽

Kotaro TAKASAKI ◽

Satoshi TAKEO ◽

Gen WATANABE ◽

Kazuyoshi TAYA ◽

...

Keyword(s):

Chorionic Gonadotropin ◽

Female Rats ◽

Immature Female ◽

Equine Chorionic Gonadotropin

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STEROL SYNTHESIS IN RATS WITH EXPERIMENTALLY-INDUCED POLYCYSTIC OVARIES

Journal of Endocrinology ◽

10.1677/joe.0.0390053 ◽

1967 ◽

Vol 39 (1) ◽

pp. 53-60 ◽

Cited By ~ 2

Author(s):

W. C. ADAMS ◽

J. H. LEATHEM

Keyword(s):

Serum Cholesterol ◽

Total Content ◽

Gonadal Hormones ◽

Female Rats ◽

Cholesterol Concentration ◽

Sterol Synthesis ◽

Sterol Metabolism ◽

Polycystic Ovaries ◽

Immature Female

SUMMARY Immature female rats were fed thiouracil for 30 days and injected with 10 i.u. human chorionic gonadotrophin (HCG) for the last 20 days. In thiouracil-fed animals, HCG produced large ovaries containing follicular cysts. These ovaries showed a subnormal concentration of cholesterol but both a normal total content and normal incorporation of [1-14C]acetate into digitonin-precipitable-sterols. Liver and serum cholesterol concentrations were reduced, but in vivo, 4 hr. incorporation of acetate into sterols was doubled suggesting either an acceleration of cholesterol turnover or delayed utilization of sterol precursors of cholesterol. HCG also reduced ovarian cholesterol concentration in euthyroid animals but total organ content and incorporation of [14C]acetate were not altered, nor were liver and serum cholesterol affected. Since the effect of induced ovarian cysts on sterol metabolism cannot be accounted for by known effects of thyroid or gonadal hormones it is suggested that influences of steroid hormones on lipid metabolism may be greatly modified in thyroid deficiency.

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Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

10.21203/rs.3.rs-18763/v5 ◽

2020 ◽

Author(s):

Kwan-Sik Min ◽

Jong-Ju Park ◽

So-Yun Lee ◽

Munkhzaya Byambaragchaa ◽

Myung-Hwa Kang

Keyword(s):

Gene Expression ◽

Chorionic Gonadotropin ◽

Expression Profiles ◽

Expression System ◽

Laboratory Animals ◽

Metabolic Clearance ◽

Qrt Pcr ◽

Significant Difference ◽

Equine Chorionic Gonadotropin

Abstract Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).Results: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.Conclusions: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

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Selective Modulation of Follicle-Stimulating Hormone Signaling Pathways with Enhancing Equine Chorionic Gonadotropin/Antibody Immune Complexes

Endocrinology ◽

10.1210/en.2009-0892 ◽

2010 ◽

Vol 151 (6) ◽

pp. 2788-2799 ◽

Cited By ~ 16

Author(s):

Vanessa Wehbi ◽

Jérémy Decourtye ◽

Vincent Piketty ◽

Guillaume Durand ◽

Eric Reiter ◽

...

Keyword(s):

Chorionic Gonadotropin ◽

Biological Activities ◽

Target Cells ◽

Pharmacological Properties ◽

Dairy Goats ◽

Glycoprotein Hormone ◽

Guanine Nucleotide Binding Protein ◽

Equine Chorionic Gonadotropin ◽

The Impact

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 αS-subunit/cAMP/protein kinase A (PKA) and β-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 β-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 β-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.

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ANTERIOR PITUITARY SENSITIVITY IN IMMATURE FEMALE RATS STIMULATED WITH GONADOTROPHIN RELEASING HORMONE IN VIVO: EFFECT OF PRIMING WITH OESTROGEN, PREGNANT MARE SERUM GONADOTROPHIN OR BRAIN LESION

Journal of Endocrinology ◽

10.1677/joe.0.0740099 ◽

1977 ◽

Vol 74 (1) ◽

pp. 99-109 ◽

Cited By ~ 3

Author(s):

D. DE ZIEGLER ◽

M. WILKINSON ◽

DANIELLE CASSARD ◽

K. B. RUF

Keyword(s):

Brain Lesion ◽

Treated Group ◽

Female Rats ◽

Similar Degree ◽

Female Rat ◽

Immature Female ◽

Releasing Hormone ◽

Gonadotrophin Releasing Hormone ◽

Pregnant Mare Serum Gonadotrophin

An investigation of pituitary sensitivity, assessed in terms of increments in plasma LH and FSH concentrations, to stimulation with one or two injections of gonadotrophin releasing hormone (GnRH) was carried out on 26-day-old immature female rats which had received one of the following priming treatments: 10 μg oestradiol benzoate (OB) as a single injection on day 23 or day 25, or on both days; 10 i.u. pregnant mare serum gonadotrophin (PMSG) on day 24; an electrochemical brain lesion placed in the mediobasal hypothalamus on day 23; control animals received either vehicle alone or a sham lesion. Pituitary sensitivity assessed at 10.00 h on day 26, after one or two injections of GnRH (100 ng/100 g body weight, s.c.), was enhanced to a similar degree in the three groups treated with OB in terms of LH (P < 0-01). The FSH response also increased after OB treatment but was not statistically significant. In contrast, 48 h after the injection of PMSG (i.e. when the rats were in a 'pro-oestrous-like' condition) pituitary sensitivity in terms of both LH and FSH dropped sharply (P < 0·001). In lesioned animals, pituitary sensitivity to one injection of GnRH was unchanged. A second GnRH injection administered after a 60 min interval induced a slightly larger LH response in control animals. In contrast, the ratio of the second response to the first increased in animals treated with PMSG, despite the state of overall decrease in sensitivity, being 4·5:1 in PMSG-treated rats versus 1·4:1 in controls. In a second set of experiments, we investigated the variation of pituitary sensitivity in conjunction with an experimentally induced gonadotrophin surge. In animals treated with OB on day 23 and with 1 mg progesterone at 12·00 h on day 26, pituitary sensitivity was increased at both 14.00 and 17.00 h as compared with that in the day 23 OB-treated group at 10.00 h. The PMSG-treated animals maintained their state of decreased responsiveness at 14.00 h, but exhibited increased pituitary sensitivity at the time of the gonadotrophin surge (17.00 h). These results show that OB increases pituitary sensitivity to GnRH in 26-day-old female rats and that the induction of a gonadotrophin surge further increases this sensitivity. In contrast, PMSG-treated rats displayed a state of decreased responsiveness 48 and 52 h, but not 55 h, after the injection. Pituitary sensitivity on the second day after PMSG treatment thus clearly differs from that observed during pro-oestrus in the adult cyclic female rat.

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Comparative gene expression profiling of mouse ovaries upon stimulation with native equine chorionic gonadotropin (eCG) and tethered recombinant-eCG

10.21203/rs.3.rs-18763/v1 ◽

2020 ◽

Author(s):

Kwan-Sik Min ◽

Jong-Ju Park ◽

So-Yun Lee ◽

Munkhzaya Byambaragchaa ◽

Myung-Hwa Kang

Keyword(s):

Gene Expression ◽

Chorionic Gonadotropin ◽

Expression Profiles ◽

Expression System ◽

Laboratory Animals ◽

Metabolic Clearance ◽

Qrt Pcr ◽

Significant Difference ◽

Equine Chorionic Gonadotropin

Abstract Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of native eCG and recombinant eCG (rec-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with native eCG and rec-eCG produced from CHO-suspension (CHO-S) cells. eCG and rec-eCG were cloned and transfected into CHO-S cells and quantified. Thereafter, we determined the metabolic clearance rate (MCR) of native eCG and rec-eCG up to 24 h after intravenous administration through the tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).Results: Rec-eCG was markedly up-regulated initially after transfection and maintained until recovery on day 9. Oligosaccharide chains were substantially modified in rec-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was slightly lower for rec-eCG than for eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentration, rec-eCG and native eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group receiving rec-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.Conclusions: The present results indicate that rec-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of rec-eCG with enhanced biological activity in vivo.

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Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

BMC Biotechnology ◽

10.1186/s12896-020-00653-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Kwan-Sik Min ◽

Jong-Ju Park ◽

So-Yun Lee ◽

Munkhzaya Byambaragchaa ◽

Myung-Hwa Kang

Keyword(s):

Gene Expression ◽

Chorionic Gonadotropin ◽

Expression Profiles ◽

Expression System ◽

Laboratory Animals ◽

Metabolic Clearance ◽

Qrt Pcr ◽

Significant Difference ◽

Equine Chorionic Gonadotropin

Abstract Background Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Results R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24 h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by > 2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses. Conclusions The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

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