• Proceedings of the KSAR Conference
• /
• 2000.10a
• /
• pp.10-12
• /
• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$ -subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• The Korean Journal of Pharmacology
• /
• v.29 no.2
• /
• pp.225-235
• /
• 1993

Pituitary LH release has been known to be regulated by the hypothalamic gonadotropin releasing hormone (GnRH) and the gonadal steroid hormones. In addition, neurotransmitters and neuropeptides are actively involved in the control of LH secretion. The alteration in LH release might reflect changes in biosynthesis and/or posttranslational processing of LH. However, little is known about the mechanism by which biosynthesis of LH subunits is regulated, especially at the level of transcription. In order to investigate if ovarian steroid hormones regulate the LH subunit gene expression, ${\alpha}\;and\;LH{\beta}$ steady state mRNA levels were determined in anterior pituitaries of ovariectomized rats. Serum LH concentrations and pituitary LH concentrations were increased markedly with time after ovariectomy. ${\alpha}\;and\;LH{\beta}$ subunit mRNA levels after ovariectomy were increased in a parallel manner with serum LH concentrations and pituitary LH contents, the rise in $LH{\beta}$ subunit mRNA levels being more prominent than the rise in ${\alpha}\;subunit$ mRNA. ${\alpha}\;and\;LH{\beta}$ subunit mRNA levels in ovariectomized rats were negatively regulated by the continuous treatment of ovarian steriod hormones for $1{\sim}4\;days$ and $LH{\beta}\;subunit$ mRNA seemed to be more sensitive to negative feedback of estradiol than progesterone. Treatment of estrogen antagonist, LY117018 or progesterone antagonist, RU486 significantly restroed LH subunit mRNA levels as well as LH release which were suppressed by estradiol or progesterone treatment. These results suggest that ovarian steroids negatively regulate the LH synthesis at the pretranslational level by modulating the steady state levels of ${\alpha}\;and\;LH{\beta}\;subunit$ mRNA and $LH{\beta}\;subunit$ mRNA seemed to be more sensitive to negative feedback action of estradiol than progesterone.

• Journal of Veterinary Clinics
• /
• v.31 no.1
• /
• pp.19-24
• /
• 2014

The purpose of the present study was to determine the priming effects of progesterone that affect the emergence of LH surge mode secretion by three different progesterone levels. In previous studies, we have shown that LH surge occurred in follicular levels of progesterone, whereas there was no surge mode secretion of LH and FSH in either the subluteal or luteal levels of progesterone. In this study, the hypothesis was that the priming effects of progesterone on the timing of the LH surge induced by exogenous estradiol are same between subluteal and luteal levels of progesterone. Long-term ovariectomized Shiba goats that had received implants of estradiol capsules (Day 0) and three different progesterone silastic packet inducing follicular, subluteal and luteal levels of progesterone were divided into three groups such as non-P, low-P and high-P group. Blood samples were collected daily throughout the experiment for the analysis of gonadal steroid hormone levels. On Day 7, all devices of progesterone packets were removed but estradiol capsules were maintained during the experiment, and blood samples were collected at 1 hr interval for 12 h from the time of progesterone removals to determine peripheral changes of estradiol and progesterone concentration. Then all animals were infused estradiol on the Day 7 after 13 h from the removals of progesterone devices with a peristaltic pump into jugular vein at a rate of 3 ${\mu}g/h$ for 36 h. For analysis of peripheral LH and estradiol concentration, blood samples were collected via another jugular vein at 2 h intervals for 52 h (from 4 h before the start of estradiol infusion to 48 h after the start of estradiol infusion). In all animals of the three groups treated with estradiol infusion, an LH surge was expressed but the peak time of LH surge was different. This time interval from estradiol infusion until the peak of LH surge was gradually and significantly extended by the different levels of progesterone treated before estradiol infusions in the three groups.

• Journal of Veterinary Clinics
• /
• v.31 no.1
• /
• pp.25-30
• /
• 2014

This study tested the hypothesis that the priming effects of progesterone on the timing of the LH surge induced by exogenous estradiol are more potentiated the negative feedback actions of progesterone on LH secretion by the existence of estradiol. In previous studies, the time interval from estradiol infusion until the peak of LH surge was gradually and significantly extended by the different levels of progesterone treated before estradiol infusions. Longterm ovariectomized Shiba goats that had received implants of estradiol capsules (Day 0) and three different progesterone silastic packet inducing follicular, subluteal and luteal levels of progesterone were divided into three groups such as non-P, low-P and high-P group. Blood samples were collected daily throughout the experiment for the analysis of gonadal steroid hormone levels. On Day 7, all devices of progesterone and estradiol packets were removed but estradiol capsules were maintained during the experiment, and blood samples were collected at 1 hr interval for 12 h from the time of progesterone removals to determine peripheral changes of estradiol and progesterone concentration. Then all animals were infused estradiol on the Day 7 after 13 h from the removals of progesterone devices with a peristaltic pump into jugular vein at a rate of 3-6 ${\mu}g/h$ for 36 h. For analysis of peripheral LH and estradiol concentration, blood samples were collected via another jugular vein at 2 h intervals for 52 h (from 4 h before the start of estradiol infusion to 48 h after the start of estradiol infusion). In all animals of the three groups treated with estradiol infusion, an LH surge was expressed but the peak time of LH surge was different. This time interval was not extended by the different levels of progesterone treated before estradiol infusions and the difference was not significant during this interval between the Low P and the High P groups. Progesterone pretreatment may contribute to regulating the neural system that is responded by estradiol, and estradiol existence potentiates the negative feedback effect of progesterone on GnRH/LH surge-generating system.

• Korean Journal of Animal Reproduction
• /
• v.24 no.4
• /
• pp.357-364
• /
• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$-subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• Applied Microscopy
• /
• v.31 no.1
• /
• pp.19-35
• /
• 2001

Outlines for plasma $estradiol-17\beta$, components, electrophoretic patterns, and ultrastructural changes were obtained in female rainbow trout (Oncorhynchus mykiss) during the seasonal reproductive cycles. Plasma $estradiol-17\beta$ under the natural conditions, exhibited distinct seasonal variation, peaking very late in vitellogenic season during September, decreasing gradually the halt of spawning in December, and ultimately falling during the early stages of seasonal ovarian recrudescence in February and March. This change in $estradiol-17\beta$ appeared to stimulate vitellogenin production as evidenced by increases in plasma calcium, phosphorus, glucose, albumin and total protein levels. The electrophoretic patterns of late maturing or spawning oocytes were stained more intensively than those of late perinucleolus oocytes (molecular weights of approximately 70,000 and 200,000). Two protein bands were found in the SDS-PAGE separation, coincident with the $estradiol-17\beta$ hormone peak. Gonadosomatic indices (GSI) significantly increased from October to January, and showed the highest peak in January, coinciding with the numerically abrupt increase of ripe ova in female. A positive correlation (r=0.701, p<0.01) was established between plasma $estradiol-17\beta$ levels and the gonadosomatic index during the prespawning. The highest level of hepatosomatic index (HSI) observed in December. During the breeding season (December), the gonadotropes were large and filled with GTH-containing inclusions such as granules and globules. The vitellogenic phase began as late perinurleolus oocytes became transformed into early maturing oocytes through the accumulation of yolk, and oocytes reached the late maturing stages as the ooplasm was completely packed with yolk. Marked ultrastructural changed in the granulosa cells during nuclear migration involve the dilation of the rough endoplasmic reticulum and the appearance of the rod-shaped mitochondria with tubular cristae. Microvilli (finger-like projections), from the zona radiata and from the oocyte grew, and made contact with each other in the pore canals of the zona radials during vitellogenesis, but were withdrawn as the zona radiata became more compact and devoid of pore canals during oocyte maturation. The zona radiata grew to a tripartite structure such as an outer thin homogeneous layer, and two inner thick helicoidal layers (zona radials interna and zona radiata externa). Under the normal conditions, the ovarian follicle influenced the histological development and periodical secretion of the hormones , sufficient for a oogenesis and gonadal steroid production.