Iron is an essential nutrient for mammalian cells. Most iron absorption occurs in the duodenal epithelial cells and is regulated by hepcidin, which is produced and secreted in the liver. High hepcidin levels can cause iron deficiency anemia due to iron absorption failure. Inside the cell, iron conjugates with a porphyrin ring and is placed with an iron coordinated to heme. One of the heme-binding proteins, known as progesterone receptor membrane component 1 (Pgrmc1), is a non-canonical progesterone receptor associated with diverse molecular gene regulation. Previous studies showed that Pgrmc1 is related to iron homeostasis via hepcidin; however, these mechanisms remain to be elucidated. In the present study, to investigate the role of Pgrmc1 in mammalian iron metabolism, we introduced Pgrmc1 knockout (KO) mice and performed molecular biological analyses using qPCR and western blotting. Pgrmc1 deficiency decreased Hamp mRNA expression and hepcidin protein levels. However, Pgrmc1 deficiency failed to decrease Hamp transcript expression and hepcidin protein levels in siPGRMC1-transfected HepG2 cells and primary Pgrmc1 KO hepatocytes, respectively. PGRMC1 knockdown cells revealed low HAMP mRNA levels upon cyclic AMP (cAMP) activation, suggesting that PGRMC1 promotes HAMP mRNA transcription via cAMP activation. It has been implicated that hepatic Pgrmc1 cannot control hepcidin directly; however, the internal environment caused by the lack of Pgrmc1 may potentially cause low hepcidin levels.

어류의 번식은 뇌에서 분비되는 다양한 신경호르몬과 뇌하수체에서 분비되는 생식소 자극 호 르몬에 의해 조절된다. 극동산 뱀장어(Anguilla japonica)의 번식도 이 호르몬들의 작용에 의해 조절되지만 성 성숙 시 신경호르몬이 뇌하수체 호르몬을 조절하는 방법은 완전히 밝혀지지 않 았다. 이전 연구에 의하면 progesterone (P4), melatonin 및 serotonin (5-HT) 등과 같은 신경호 르몬이 일부 어류의 번식 과정 조절에 관여하는 것으로 밝혀졌다. 본 연구에서는 뱀장어의 뇌 하수체를 초대 배양하였고, 안정화된 뇌하수체 세포에 P4, 17β-estradiol (E2), melatonin 및 5- HT를 처리하였다. 이후 처리된 호르몬의 작용이 뇌하수체 세포에서 번식 관련 호르몬인 FSHβ, LHβ, GH 및 SL mRNA 발현에 어떤 영향을 미치는지 조사하였다. 본 연구를 수행한 결과, P4는 뇌하수체 세포에서 FSHβ와 LHβ 발현을 증가시켰고, melatonin은 FSHβ와 LHβ 뿐만 아니라 GH 와 SL의 발현을 증가시켰다. 하지만 5-HT는 이 유전자의 mRNA 발현에 유의한 영향을 미치지 않았다. 이상의 결과는 P4 또는 melatonin이 뱀장어의 초기 성 성숙에 중요한 역할을 할 수 있음을 의미한다.

This study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.

Carcass grade primarily depends on marbling of intramuscular fat, which is associated with the texture and tenderness of beef. Accordingly, various economical molecular tests for high intramuscular fat in beef have been attempted. Especially, Hanwoo (Korean Cattle) intramuscular fat has higher levels of monounsaturated fatty acids than that in the beef of other cattle. Intramuscular fats are associated with levels of lipid metabolic genes in the liver transcriptome. Therefore, hepatic triglyceride synthesis can considerably increase intramuscular fat. To investigate the relationship between hepatic lipogenesis and carcass grade, we analyzed 52 Hanwoo liver samples from domestic farms, and evaluated lipid levels and transcript levels of glucose and lipid metabolism-related genes according to carcass grade. Oil-Red-O staining revealed fatty livers in high carcass grades. Moreover, we found significantly higher levels of mRNA for lipogenesis, glycolysis, and triglyceride synthesis genes in high carcass grade livers. Importantly, progesterone receptor membrane component 1 (Pgrmc1) levels were significantly lower in high carcass grade livers. As Pgrmc1 suppression is correlated with induction of de novo lipogenesis (DNL) and glycolysis genes, it has a diagnostic impact for high carcass grades. These results could be used for genetic improvements in carcass grades of cattle. More importantly, as Pgrmc1 can be detected in blood peripheral nucleated cells, it also has value for rapid blood diagnosis.

One of the major hallmarks of uterine diseases is disruption of ovarian steroid hormone control of uterine cell proliferation and differentiation. Estrogen (E2) stimulates proliferation of uterine epithelial cells while progesterone (P4) is inhibitory to E2-mediated proliferation of the epithelium. Mitogen inducible gene 6 (Mig-6) is an important mediator of P4 signaling to inhibit E2 signaling in the uterus. Uterine-specific knockout of Mig-6 caused endometrial P4 resistance and infertility. Levels of ErbB2 (also known as HER2) and phospho-ERK1/2 are significantly higher in Mig-6 knockout mice as well as infertile women with endometriosis. To determine the interplay between Mig-6 and the Erbb2 signaling pathway in the uterus, we generated mice with Mig-6 and Erbb2 conditionally ablated in progesterone receptor-positive cells (Pgrcre/+ Mig-6f/f Erbb2f/f; Mig-6d/d Erbb2d/d). Mig-6d/d mice were infertile whereas control and Mig-6d/d Erbb2d/d mice exhibited normal fecundity. The uterine horns of Mig-6d/d mice had no implantation sites, whereas control and Mig-6d/d Erbb2d/d mice had averaged implantation sites. Additionally, aberrant increment of epithelial proliferation in uterus of Mig-6d/d mice did not show in Mig-6d/d Erbb2d/d mice uterus at pre-implantation stage. Microarray analysis revealed that almost altered genes in Mig-6d/d mice were recovered their expression levels in Mig-6d/d Erbb2d/d mice. The altered pathways such as cell-cycle control, DNA replication, and modification processes by Mig-6 ablation were rescued in Mig-6d/d Erbb2d/d mice. The infertility seen in Mig-6d/d mice is recovered in Mig-6d/d Erbb2d/d mice. These results suggest that Mig-6 mediates a critical P4 function to inhibit E2 signaling by inhibiting ErbB2 signaling. As MIG-6 is a mediator of P4 signaling, the activity of which can suppress unopposed-E2 signaling, our studies provide a potential new drug target for the intervention of female infertility.

Progesterone regulates endometrial functions to support implantation, placentation, and fetal/placental development in the uterus. It is known that actions of progesterone are mediated by nuclear progesterone receptor (PGR), using the signaling pathway referred to the genomic pathway. However, all physiological progesterone actions cannot be explained by the genomic pathway via PGR, and it is understood that there are non-genomic actions of progesterone though membrane progesterone receptors, progesterone receptor membrane components (PGRMCs) and progestin and adipoQ receptors (PAQRs). The expression and localization of PGRMCs and PAQRs has been reported in female reproductive tissues of several species such as human, mouse and cattle. Previously, we have shown that PGRMCs and PAQRs are expressed in the porcine uterine endometrium during the estrous cycle and pregnancy. However, the regulatory mechanism for expression of PGRMCs and PAQRs in the uterine endometrium has not been studied in pigs. Thus, to understand the regulatory mechanism of PGRMC1, PGRMC2, PAQR5, PAQR6, PAQR7, PAQR8, and PAQR9 expression in the uterine endometrium during the estrous cycle and pregnancy in pigs, we determined the effect of steroid hormones estrogen and progesterone on expression of PGRMCs and PAQRs using the endometrial tissue explants for immature pigs. Levels of PGRMC1, PGRMC2, PAQR5, PAQR6, PAQR7, PAQR8, and PAQR9 mRNAs were increased by increasing doses of progesterone, but not by estradiol in the uterine endometrium. Blocking PGR by treatment of RU486, a progesterone receptor antagonist, increased levels of endometrial PGRMCs and PAQRs mRNA. These data showed that membrane progesterone receptors were induced by progesterone in the uterine endometrium, suggesting that these membrane progesterone receptors may play an important role in mediating progesterone actions in the uterine endometrium for regulation of the estrous cyclicity and pregnancy. [Supported by the Next Generation Biogreen 21 Program (# PJ01119103), Rural Development Administration, Republic of Korea]

This study was to investigate effect of progesterone (P4) on prostaglandin (PG) synthases and plasminogen activators (PAs) system in bovine endometrium during estrous cycle. Endometrium tissues were collected from bovine uterus on follicular and luteal phase and were incubated with culture medium containing 0 (Control), 0.2, 2, 20 and 200 ng/ml P4 for 24 h. The PGF2α synthase (PGFS), PGE2 synthase (PGES), cyclooxygenase-2 (COX-2), urokinase PA (uPA), and PA inhibitors 1 (PAI-1) mRNA in bovine endometrium were analyzed using reverse transcription PCR and PA activity was measured using spectrophotometry. In results, COX-2 was higher at 2 ng/ml P4 group than control group in luteal phase (p<0.05), but, it did not change in follicular phase. Contrastively, PGES was significantly increased in 2 ng/ml P4 group compared to control group in follicular phase, but there were no significant differ among the treatments in luteal phase. uPA was no significant difference between P4 treatment groups and control group in both of different phase. PAI-1 was decreased in 20 ng/ml P4 group compared to control group in follicular phase (p<0.05). PA activity was decreased in 2 ng/ml P4 group compared to other groups in follicular and luteal phase (p<0.05). In conclusion, we suggest that P4 may influence to translation and post-translation process of PG production and PA activation in bovine endometrium.

A study was carried out on 16 indigenous ewes in Bangladesh in order to assess the reproductive physiology, the pattern of vaginal cell exfoliation and progesterone profiles during the estrous cycle period. The mean estrous cycle length and duration of estrus were 15.8±0.12 days and 31.1±0.57 h respectively. The exfoliated epithelial cells were categorized into parabasal, intermediate, superficial and keratinized and their relative occurrences. The percentages of parabasal, intermediate and superficial cell type during proestrus were similar. The percentage of superficial cell type during estrus was 61.7%, which was significantly (p<0.01) differ from other types of cells and stages of estrus cycle. Metoestrus was predominant with neutrophils in addition with other cell types. Dioestrus was dominated by neutrophils. On days 0 to 5 of the cycle the progesterone concentration was 0.09 to 1.6±0.07 ng/ml. The length of diestrus was 5∼10 days with a range of mean progesterone level of 1.6±0.07 to 2.8±0.11 ng/ml. Progesterone levels increased significantly (p<0.01) after Day 5 and maximum level was 2.8±0.11 ng/ml observed on Day 10 of the estrous cycle. Thereafter it dropped rapidly to basal level of 0.11±0.04 ng/ml on Day 0 (p<0.01). These results indicate that the pattern of exfoliation of vaginal cells along with progesterone concentration could be used to determine the reproductive stages of indigenous ewe.

This study was conducted to investigate the changes of hormone levels of serum progesterone (P4) and estradiol-17 β (E2) in sows of Landrace (L), Yorkshire (Y) and F1 (L × Y) (respectively n=3) with excellent ability, and to provide a baseline data for improving reproductive performance. In this experiment, the sows at the age of 12 months or more were used. The sows were fed by two way methods, one is conventional methods and the other is 3 days-flushing feed before estrus. Each pig’s blood was collected in 3, 6, 9, 12, 15 and 18 days after the estrus for the analyses of P4 and E2. Serum was separated by centrifugation for 15 min. with 3,000 rpm. Progesterone and estradiol-17β were measured by immunochemical assay (ELIZA test). In conventional feeding, serum progesterone levels were significantly (p<0.01) higher in F1 than in L and Y. No significant differences in P4 concentrations were seen between the L and Y of sows. Serum E2 levels were similar the serum progesterone levels. In the case of flushing feed, the tendency of hormonal changes were similar to conventional methods. But almost of hormonal levels were a little higher than that of conventional methods. P4 level of L and Y in flushing feed were significantly different (p<0.01). Serum E2 level of Y in flushing feed was significantly different among the breeds (p<0.01). These results were similar to the tendency of hormonal changes in general sows and moreover, flushing feed is known to develop the swine production, these results proved the fact of the methods. And these results suggested that more studies about hormonal changes in sows according to seasonal and nutritional factors should be needed.

It is generally accepted that chronic stress impairs female reproduction. It negatively affects ovarian function and the number of ovulated oocytes. Chronic stress lowers the number of retrieved oocytes. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. The main corticosteroids are cortisol, cortisone, 11-deoxycortisol and corticosterone, cortisol being one of the most commonly used welfare and stress physiological indicator. In this study, we investigated the effect of cortisol level on progesterone patterns and ovulation in the dog. Cortisol and progesterone level of serum were analyzed by radioimmunoassay. The day of ovulation was considered as the day when serum progesterone concentration was 6.0∼8.0 ng/ml. In vivo dog oocytes were collected by flushing oviducts of mixed-breed bitches at three days after ovulation. We classified dogs as having group 1 (cortisol level, 0 ≤ or < 2 μg/dl), group 2 (corisol level, 2 ≤ or < 4 μg/dl), group 3 (cortisol level, 4 ≤ or < 6 μg/dl) and group 4 (cortisol level, 6 μg/dL ≤). The patterns of progesterone were not different in four cortisol groups. The average numbers of retrieved oocytes was not different in four cortisol groups. These results suggest that different cortisol levels on estrus dogs do not affect ovulation, number of ovulated oocytes and progesterone changes.

The objective of this experiment was to assess the relationship between electrical resistance of the vaginal mucosa and plasma progesterone for optimal mating time in the bitch. Eight mature beagle bitches were examined, and we observed eight times of estrus. Vaginal electric resistance was recorded weekly using a Draminski ovulation detector in anestrus, and daily in estrus. Plasma progesterone concentration was estimated by radioimmunoassay. In the bitch, incline in vaginal electric resistance (376.20 ± 105.63 units) showed a closely association with the onset of proestrus. Ovulation day was determined as the first day when plasma progesterone concentration increased above 5.0 ng/ml (Day 0). On Day 0, vaginal mucous electric resistance was 438 ± 132 units. Vaginal mucous electric resistance showed a slight decrease or was maintained until Day 0. However, it showed an explosive increase, and peaked on Day 1~3, which was above 600 units. Two of eight cases peaked on Day 1, three of eight cases were revealed on Day 2, and others were revealed on Day 3. After Day 4, resistance showed a rapid drop to below 600 units and reached 200 units on Day 8. The optimal mating time was determined when vaginal mucous electric resistance was above 600 units.

The purpose of the present study was to investigate the effect of IFN- on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and concentration of progesterone (P4) in endometrial cells. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses of IFN- (0, 0.02, 0.2 and 2 ug/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin and levels in the culture media were analyzed by enzyme immunoassays and total RNA was extracted from the cells for RT-PCR. P4 concentrations of blood samples were assayed by chemiluminescent immuno assays system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN- (p<0.05), but it was not significantly different in all groups of stromal cells except for 2 ug/ml IFN- group (p<0.05). Although IFN- did not affect and production in epithelial cells, it decreased and production significantly in stromal cells (p<0.05). In vivo experiment, blood concentration of P4 was significantly increased after addition of IFN- (1 ug/ml). The results indicate that PG production was mediated by COX-2 expression in stromal cells but it was not affected in epithelial cells and this suggest that treatment of IFN- could improve the implantation environment of uterine by maintenance of high P4 concentration.

In ruminants, Interferon-τ (IFN-τ) has the role of recognizing pregnancy signals produced by the embryo and it may have an important role during the luteolysis. Therefore, the purpose of the present study was to investigate the effect of IFN-τ on prostaglandin synthesis, cyclooxygenase-2 (COX-2) gene expression in vitro and secretion of progesterone (P4) in vivo. The epithelial and stromal cells isolated from bovine endometrium were cultured with different doses of IFN-τ (0, 0.02, 0.2 and 2 μg/ml). Human chorionic gonadotropin (hCG, 1.5 IU/ml) was used as a positive control. Prostaglandin E2 and F2α levels in the culture media were analyzed by enzyme immunoassays, and total RNA was extracted from the cells for RT-PCR. P4 concentrations in blood samples were assayed by chemiluminescent immunoassay system. In epithelial cells, COX-2 gene expression was increased in the presence of IFN-τ (p<0.05), but it was not significantly different in all groups of stromal cells except 2 μg/ml IFN-τ group (p<0.05). Although IFN-τ did not affect PGE2 and PGF2α production in epithelial cells, it decreased PGE2 and PGF2α production significantly in stromal cells (p<0.05). In vivo experiment, the P4 concentrations in blood sample was significantly increased after injection of 1 μg/ml IFN-τ. These results indicate that PG production was mediated by COX-2 expression in the stromal cells but it did not affect in the epithelial cells, and suggest that treatment of IFN-τ was to improve the implantation environment of uterine by maintenance of high P4 concentration. * This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ907008)” Rural Development Administration, Republic of Korea.

Growth hormone (GH) is obligatory for growth and development. But, there is controversy on the GH effect about reproductive processes of sexual differentiation, pubertal maturation, gonadal steroidogenesis, gametogenesis and ovulation. This study was conducted to investigate the effect of GH on estrus, ovulation and embryo implantation. The results obtained were as follows. GH stimulated to increase estrus rate (p<0.05), pregnancy rate (p<0.05), and total fetus number in mice treated for superovulation. Also, the correlation between GH and steroids, E2 and P4, at peri-estrus stage/ peri-ovulation stage/ peri-implantation stage of the superovulation-induced mice was examined. Consequently, GH co-injected with PMSG especially increased P4 level (p<0.05) at peri-estrus stage of superovulationinduced mice. In conclusion, GH co-treatment in superovulation system boosted the rate of estrus, pregnancy and total fetus by increasing progesterone level at peri-estrus stage of superovulation-induced mice.

Acyl-CoA synthetase 4(ACSL4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. The human and mouse ACSL4 gene are mapped on chromosome X. The female mice heterozygous for ACSL4 deficiency became pregnant less frequently and produced small litters, with 40% of embryos surviving gestation. In this study, we examined the regulation of ACS4 by estradiol-17β and progesterone (P4) in the human endometrial cancer cell line HTB-1B. ACSL4 mRNA was increased in a dose-dependent manner. Also, expression of ACSL4 gene was up-regulated in a time-dependent manner in HTB-1B cells. However, combined treatment with progesterone and estradiol-17β modestly decreased the levels of ACS4L mRNA as compared with the estradiol-17β and progesterone respectively. Overall, these results suggest that the ACSL4 gene is regulated by progesterone and estradiol-17β in the HTB-1B cells.

Ovulation synchronization (ovsynch) has proved to increase the number of insemination in cattle by overcoming the problems of heat detection. The aim of this study was to do ovsynch in water buffaloes where heat detection is a major reproductive problem and to determine the conception rates after timed artificial insemination (TAI). Twenty cyclic buffaloes at 60 days postpartum were selected by examining 24 unobserved estrus buffaloes based on milk progesterone assay (progesterone concentration 1.0 ng/ml) from the Mymensingh district of Bangladesh. Ovsynch treatment regimen was started irrespective of the stage of estrous cycle. Gonadorelin (500 ) was injected intramuscularly at Day 0 followed by Alfaprostol (8 mg) at Day 7. A second injection of Gonadorelin was given at Day 9 and TAI was done with frozen semen from Mediterranean buffalo bulls at 16~20 hours of the second Gonadorelin injection. Milk progesterone ELISA at Day 10~12 post AI confirmed ovulation in 16 out of 20 (80%) buffaloes (progesterone concentration 1.0 ng/ml). High progesterone concentration ( 1.0 ng/ml) at Day 10~12 and Day 22~24 of AI showed pregnancy in six out of 20 (30%) buffaloes. Pregnancy was further confirmed by ultrasonography at Day 40 in these six buffaloes. In conclusion, ovsynch followed by TAI could be applied in cyclic buffaloes for overcoming the estrus detection problems; however, more studies are needed to increase the conception rate.

This study investigated potential relationship between fetal deaths and plasma progesterone concentrations of bitches. Serial ultrasonographic examinations were performed on small-pet 23 dogs from gestation day (GD) 15 through parturition. The dogs were 3 non-pregnant bitches, 9 spontaneous delivery bitches, 6 partial early embryonic death bitches, 2 whole early embryonic death bitches, and 3 aborted bitches. The late pregnancy (GD 51-54) appeared in 2 of the 3 aborted bitches and the hypoluteoidism appeared in 1 of the 3 aborted bitches. The plasma progesterone concentrations of partial early embryonic death bitches (n=6) showed no significant difference when compared with the spontaneous delivery bitches. We observed that plasma progesterone concentrations were dramatic decrease before the onset of embryonic death in whole early embryonic death bitches that plasma progesterone concentrations of aborted bitches at late pregnancy were significantly decreased when compared with those of spontaneous delivery bitches. The plasma progesterone concentrations of the hypoluteoidism bitch were lower than those of spontaneous delivery bitches. At the hypoluteoidism bitch, fetuses were resorbed in early pregnancy and aborted in late pregnancy. On the basis of the results, the diagnosis of partial early embryonic death could not be confirmed without ultrasonographic examination. The partial early embryonic death was considered a spontaneous phenomenon and uncorrelated with plasma progesterone concentration. However, aborted bitches and whole early embryonic death bitches were related to plasma progesterone concentrations and that of bitches gradually decreased before fetal death. These findings suggest that administration of progesterone may be a useful preventing agent against fetal death.

Most captive canids and felids at Zoos in advanced countries have been examined enough to apply artificial reproductive techniques to them. We investigated reproductive hormones and vaginal epithelial cells of a 6-year-old, female coyote, hoping these data could eventually be extended to artificial insemination with frozen-thawed conspecific semen at Seoul Zoo. As a relative of pet dogs, coyote exhibited a similar appearance with only minor differences. In vaginal smear, an increase in the number of superficial cells suggests that the bitch has reached a state close to estrus. A sudden decrease of estradiol and increase of progesterone is considered as a preovulatory event. Vaginal epithelial cells and hormones might be useful for determining the optimal time of artificial insemination in coyotes' breeding.