Regulation Of LH Research Articles

SAT608 Gonadotrope-specific Deletion Of Icer Results In Increased Lh Pulsatility But Reduced Fsh Levels In Mice

Abstract Disclosure: N.P. Boris: None. S.A. Pereira: None. G.A. Stamatiades: None. K.N. Hausken: None. H. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Background: The hypothalamic-pituitary-gonadal (HPG) axis is regulated by pulsatile secretion of hypothalamic GnRH, which acts on anterior pituitary gonadotropes to control synthesis and secretion of LH and FSH. High and low GnRH pulse frequencies preferentially stimulate Lhb and Fshb expression, respectively, through mechanisms that are not completely understood. The mouse Fshb gene promoter contains a half-AP-1/half-cAMP response element (CRE) motif; we have shown previously that this element is bound by CRE-binding protein (Creb) in vitro to increase expression in response to low GnRH pulse frequencies, but is antagonized by inducible cAMP early repressor (Icer) at higher GnRH pulse frequencies, resulting in a lower level of Fshb transcription. In this study, we aimed to identify the role of Icer in gonadotropin regulation in vivo in mice. We hypothesized that gonadotrope-specific deletion of Icer may influence pubertal timing and fertility due to an increase in Fshb expression. Methods and Results: Gonadotrope-specific deletion of Icer (GnrhrGRIC/+/Icerfl/fl; Icer KO) and control (Icerfl/fl) mice were generated, and pubertal and reproductive phenotypes were evaluated. There was no difference in body weight or in pubertal timing, evaluated by age of preputial separation in males and by vaginal opening and first estrus in females. In post-pubertal male mice, neither basal LH and FSH levels nor the LH or FSH response to acute GnRH stimulation were significantly different in Icer KO mice, compared to littermate Icerfl/fl controls. Male Icer KO mice 14 days post-gonadectomy (GDX) had significantly more LH pulses within a three-hour period than controls (Icer KO 5.83 ± 0.4 pulses vs Icerfl/fl 4.66 ± 0.3 pulses; p=0.049), but no differences in basal LH or LH pulse amplitude. Interestingly, serum FSH levels were significantly lower in Icer KO mice compared to Icerfl/fl controls 2 days post-GDX (Icer KO 20.0 ± 1.0 vs Icerfl/fl 26.0 ± 0.8; p=0.003) and 7 days post-GDX (Icer KO 18.0 ± 0.4 vs Icerfl/fl 22.5 ± 0.8, p=0.002). There were no significant differences in pituitary Fshb, Lhb, or Gnrhr mRNA levels between Icer KO and Icerfl/fl control mice 14 days post-GDX. Conclusions: Deletion of Icer from pituitary gonadotropes in mice did not affect body weight or timing of pubertal onset. However, in GDX male mice, LH pulse frequency was increased, whereas the post-GDX increase in serum FSH levels was blunted, in Icer KO mice compared to controls. These findings support a role for Icer in both LH and FSH regulation in vivo and highlight the importance of extending in vitro observations to in vivo models. Presentation: Saturday, June 17, 2023

Utilization of water lily activated charcoal as a biofilter in tofu wastewater treatment

The purpose of this study was to determine the effect of water lily activated charcoal as a biofilter in tofu wastewater treatment and to determine the effective dose of water lily activated charcoal as a biofilter in tofu wastewater treatment. The results showed that water lily activated charcoal can be used to improve the quality of tofu liquid waste; thus, it can improve the quality of tofu liquid waste which can be seen from the parameters of TSS (Total Suspended Solid). The treatment dose of 2 g of activated charcoal (A3) is the best dose to improve the quality of tofu liquid waste and has complied with the 2014 LH regulation concerning the quality standards of wastewater for the tofu industry, namely the parameters of TSS. The existence of this research can be a reference for further similar research by utilizing organic matter as an alternative in tofu wastewater treatment.

Characterization of the pars tuberalis and hypothalamus transcriptome in female sheep under different reproductive stages

For understanding the molecular events underlying the follicular (F) and luteal (L) phases of estrous cycle, and anestrous (A) phase, the pars tuberalis (PT), and hypothalamus (HT) transcriptomes of 21 ewes were studied. In HT, 72 and 3 differential expression genes (DEGs) were found when comparing F vs. A and L vs. A, respectively. In PT, 6 and 4 DEGs were found in F vs. A and L vs. A comparisons, respectively. Enrichment analysis for DEGs between the F and A phases in the HT revealed significant clusters, mainly associated with actin-binding, and cytoskeleton, that are related to neural plasticity modulated by gonadal steroid hormones, as well as with oxytocin signaling. We found that DEGs in PT had higher differences in expression levels than those found in HT. In this sense, the ITLN was highly upregulated in the F and L vs. A phases, being MRPL57 and IRX4 highly downregulated in L vs. A comparison. The DDC gene in PT, related to LH regulation, was upregulated in the F phase. The gene set enrichment analysis (GSEA) revealed multiple pathways related to neurotransmission and neuronal plasticity. Our study reveals new candidate genes involved in the reproductive stages’ transitions in seasonal sheep.

Neuroendocrine mechanisms underlying estrogen positive feedback and the LH surge.

A fundamental principle in reproductive neuroendocrinology is sex steroid feedback: steroid hormones secreted by the gonads circulate back to the brain to regulate the neural circuits governing the reproductive neuroendocrine axis. These regulatory feedback loops ultimately act to modulate gonadotropin-releasing hormone (GnRH) secretion, thereby affecting gonadotropin secretion from the anterior pituitary. In females, rising estradiol (E2) during the middle of the menstrual (or estrous) cycle paradoxically “switch” from being inhibitory on GnRH secretion (“negative feedback”) to stimulating GnRH release (“positive feedback”), resulting in a surge in GnRH secretion and a downstream LH surge that triggers ovulation. While upstream neural afferents of GnRH neurons, including kisspeptin neurons in the rostral hypothalamus, are proposed as critical loci of E2 feedback action, the underlying mechanisms governing the shift between E2 negative and positive feedback are still poorly understood. Indeed, the precise cell targets, neural signaling factors and receptors, hormonal pathways, and molecular mechanisms by which ovarian-derived E2 indirectly stimulates GnRH surge secretion remain incompletely known. In many species, there is also a circadian component to the LH surge, restricting its occurrence to specific times of day, but how the circadian clock interacts with endocrine signals to ultimately time LH surge generation also remains a major gap in knowledge. Here, we focus on classic and recent data from rodent models and discuss the consensus knowledge of the neural players, including kisspeptin, the suprachiasmatic nucleus, and glia, as well as endocrine players, including estradiol and progesterone, in the complex regulation and generation of E2-induced LH surges in females.

Effects and related mechanisms of Huang's SiYu pills on Leydig cell function in kidney-yin deficiency rat models

The prescription “Huang's SiYu Pill” (HSYP) was founded by professor Huang Haibo, a famous traditional Chinese medicine in Inner Mongolia, based on long-term clinical experience. In this study we observed the effects of HYSP on sperm quality, hormone level and the expression level of reproductive related genes and proteins in testicular stromal cells of rats with kidney-yin deficiency to explore the therapeutic mechanism of HSYP in the treatment of infertility with kidney-yin deficiency, Liuwei Dihuang Pill group (LDP) and Tamoxifen Citrate Tablets combined with Pancreatic Kallidinogenase Enteric Coated Tablets were used to be the positive control drug. After 25 days of medicine administration, the animals were anesthetized and killed, the serum and testicular tissues were retained for further experiments. The ELISA results showed that HSYP had a better effect on increasing serum FSH level than the conventional Chinese medicine or western medicine, while there was no remarkable difference in the regulation of serum LH in each administration group. The results of RT-qPCR showed that the transcription levels of Cyp11a1, Hsd3b1, Lhcgr and Star genes in the medium and high dose groups of HSYP were significantly upregulated compared with the model group of kidney-yin deficiency, while the expression of Nr5a1 gene was not significantly affected. The results of HE and immunohistochemistry showed that HSYP significantly improved the development level of testicular stromal cells and promoted the expression levels of CYP11A1 and HSD11B1 in testicular stromal cells, compared with the model group of kidney-yin deficiency. Together, our results showed that Huang's SiYu Pills can significantly improve the development and function of Leydig cells in SD rats with kidney-yin deficiency model, which provided a good research basis for deep research on relevant pharmacological mechanisms of the medicine.

Evaluasi Teknik Operasional dari Kinerja Instalasi Pengolahan Lumpur Tinja di Kota Kendari

Fecal waste requires treatment (IPLT) that contains pollutant compounds which result in creating damage to the environment or potentially creating pollution. Therefore, we need a performance evaluation to optimize the performance of the Kendari City IPLT. The Kendari City IPLT serves processing of sewage sludge throughout the sub-district with sources from domestic waste processing units and facilities. Sludge desludging is only carried out by 3% of the total septic tank units that must be vacuumed. The purpose of this study is to determine the effectiveness of the performance of the IPLT in treating waste and determine whether the effluent generated from the IPLT has met the specified quality standards. This research method is to compare the effectiveness of Kendari City IPLT conditions with LH Regulation No. 05 of 2014. The BOD content after analysis was 125.1 mg/L and the oil content was 1 mg / l. The Kendari City IPLT treatment unit consisting of an imhoff tank, anaerobic pool, facultative pool and maturation pool in good condition some ponds did not meet the criteria according to the Ministry of Public Works in 1998. The imhoff tank was only 3.7 m (criteria 6-9 m), the anaerobic pool caused odor that can interfere, facultative pool water height of 2.0 m (criteria 1.2 - 2.4 m).

Evaluasi Teknik Operasional dari Kinerja Instalasi Pengolahan Lumpur Tinja di Kota Kendari

Fecal waste requires treatment (IPLT) that contains pollutant compounds which result in creating damage to the environment or potentially creating pollution. Therefore, we need a performance evaluation to optimize the performance of the Kendari City IPLT. The Kendari City IPLT serves processing of sewage sludge throughout the sub-district with sources from domestic waste processing units and facilities. Sludge desludging is only carried out by 3% of the total septic tank units that must be vacuumed. The purpose of this study is to determine the effectiveness of the performance of the IPLT in treating waste and determine whether the effluent generated from the IPLT has met the specified quality standards. This research method is to compare the effectiveness of Kendari City IPLT conditions with LH Regulation No. 05 of 2014. The BOD content after analysis was 125.1 mg/L and the oil content was 1 mg / l. The Kendari City IPLT treatment unit consisting of an imhoff tank, anaerobic pool, facultative pool and maturation pool in good condition some ponds did not meet the criteria according to the Ministry of Public Works in 1998. The imhoff tank was only 3.7 m (criteria 6-9 m), the anaerobic pool caused odor that can interfere, facultative pool water height of 2.0 m (criteria 1.2 - 2.4 m).

Transcriptome of Distinct LH and FSH Cells Reveals Different Regulation Unique to Each Cell Type

From mammals to fish, gametogenesis and sexual maturation are driven by LH and FSH, the two gonadotropic hormones temporally secreted from the pituitary. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin hormone since, unlike mammals; they synthesize and secrete LH and FSH from distinct cells. By performing cell specific transcriptome analysis of double-labelled transgenic Nile tilapia (Oreochromis niloticus) expressing GFP and RFP in LH or FSH cells, respectively, we identified genes specifically enriched in each cell type. Though GnRH is considered the main neuropeptide regulating LH and FSH, we found that each LH and FSH cell express unique GPCR signature that reveals the direct regulation of additional metabolic and homeostatic hormones (like cholecystokinin, somatostatin and glutamate). Moreover, some of those GPCRs were conserved also in gonadotrophs of mammals (like PACAP receptor, Adropin receptor and GABBA receptor). Next, we had exploited the unique behavior of Nile tilapia where a behavioral hierarchy is created between males, to compare the gene expression in the pituitary and brain of dominant (reproducing) males to a subordinate (non-reproducing) males. By combining the two transcriptome sets we had identified novel players in the hypothalamic regulation of the HPG axis, and revealed how brain aromatase (cyp19a1b), that is enriched specifically in LH cells, is the key factor in regulating the activity of LH and FSH cells in dominant reproducing fish. Thereby, unraveling novel mechanisms in the differential regulation of LH and FSH. The research was funded by the Israel Science Foundation (ISF) no. 1540/17.

Oxytocin-like signal regulates Lh cells directly but not Fsh cells in the ricefield eel Monopterus albus†.

The synthesis and release of LH and FSH in the pituitary of vertebrates are differentially regulated during gonadal development and maturation. However, the underlying neuroendocrine mechanisms remain to be fully elucidated. The present study examined the possible involvement of isotocin (Ist), an oxytocin-like neuropeptide, in the regulation of Lh and Fsh in a teleost, the ricefield eel Monopterus albus. The immunoreactive isotocin receptor 2 (Istr2) was shown to be localized to Lh but not Fsh cells. In contrast, immunoreactive isotocin receptor 1 (Istr1) was not observed in either Lh or Fsh cells in the pituitary. Interestingly, Lh cells in female ricefield eels expressed Istr2 and secreted Lh in response to Ist challenge stage-dependently and in correlation with ovarian vitellogenesis. Moreover, Ist decreased Lh contents in the pituitary of female fish, indicating its stimulatory roles on Lh release in vivo. The induction of Lh release by Ist in dispersed pituitary cells was blocked by a PLC or IP3R inhibitor but not by a PKA or PKC inhibitor, indicating the involvement of the IP3/Ca2+ pathway. Collectively, the above results indicate that isotocin may bind to Istr2 to stimulate Lh release via the IP3/Ca2+ pathway, and play important roles in the ovarian maturation in ricefield eels. Furthermore, the present study suggests a novel neuroendocrine mechanism underlying the differential regulation of Lh and Fsh in vertebrates.

Nutritional regulators of the hypothalamic–pituitary axis in pigs

Nutritional signals are detected by the central nervous system (CNS) and translated by the neuroendocrine system into signals that alter secretion of LH and growth hormone (GH). Furthermore, these signals directly affect the activity of the pituitary gland independently of CNS input. Insulin-like growth factor I (IGF-I), insulin, leptin and specific metabolites, such as glucose and free fatty acids (FFA)', are potential signals of the metabolic status to the brain-pituitary axis. Intravenous injection of a lipid emulsion or glucose suppressed the GH and LH response to GH releasing hormone (GHRH) and GnRH, respectively. Insulin and IGF-I regulation of LH and GH secretion occur at the pituitary gland. Feed deprivation for 24 h suppressed leptin secretion without affecting LH or GH secretion, whereas central administration of leptin resulted in a decrease in feed intake and an increase in GH secretion. Oestrogen-induced leptin gene expression in adipose tissue increased with age and adiposity in pigs. Leptin stimulated GnRH release from hypothalamic tissue in vitro. These results identify putative signals that link metabolic status and neuroendocrine control of growth and reproduction by altering endocrine function during periods of fasting, feed restriction and lactation.

Model of Motor Vehicle Gas Distribution Based on Ecology- Health, Economic, Social-Cultural and Law Factors in the City of Pekanbaru

Air pollution will make the city environment unhealthy and can interfere with human health, therefore one must strive to not increase air pollution. One way to reduce air pollution in cities is to reduce carbon emissions and build Green Open Spaces (GOS). Therefore, the purpose of this study is to create a model for distribution of motor vehicle exhaust emissions in the city of Pekanbaru. Air pollution at a certain level can be a combination of one or more pollutants, either in the form of solids, liquids or incoming gases dispersed into the air and then spread to the surrounding environment. Further analysis of environmental factors in the form of socio-cultural, economic and ecological factors are explored in this study. Other important environmental parameters in pollutant studies are ecological factors as well. While the economic factors in question is the willingness to pay, it shows the minimum cost needed to anticipate the amount of exhaust emissions caused by motorized vehicles and calculate the economic value associated with public health. The emission of gas produced by each vehicle is below the predetermined standard quality threshold, namely LH Regulation No. 12 of 2010. Although the measurement results in Table 1 do not exceed ambient air quality standards, but the increase in a trend that can cause air quality deterioration was detected. The components in these two strategies need to get more emphasis and attention so that the existence of charcoal trading business can be sustainable. The results obtained from this study are a model of exhaust gas distribution in the form of gas emission distribution contours supported by ecological data (E), economics (E) and socio-cultural (S), especially in creating recommendation models in policy making both in the form of laws, government regulations or regional regulations, which is the simplest prerequisite for motor vehicle owners when the vehicle registration is extended. This model is better known as the E2S + H model.

Pituitary Action of E2 in Prepubertal Grass Carp: Receptor Specificity and Signal Transduction for Luteinizing Hormone and Follicle-Stimulating Hormone Regulation.

17β-estradiol (E2) is an important sex steroid produced by ovary and brain. In mammals, E2 plays an important role in hypothalamus–pituitary–gonad axis to regulate puberty onset, however, little is known about the functional role of E2 in teleost pituitary. Using prepubertal grass carp as model, three nuclear estrogen receptors (nERs: estrogen receptor alpha, estrogen receptor beta 1, and estrogen receptor beta 2) and two G protein-coupled estrogen receptors (GPER1: GPER1a and GPER1b) were isolated from grass carp pituitary. Tissue distribution analysis indicated that both nERs and GPERs were highly detected in grass carp pituitary, which suggested that E2 should play an important role in grass carp pituitary. Using primary cultured grass carp pituitary cells as model, high-throughput RNA-seq was used to examine the E2-induced differentially expressed genes (DEGs). Transcriptomic analysis showed that E2 could significantly upregulate the expression of 28 genes in grass carp pituitary cells, which were characterized into different functions including reproduction, gonad development, and central nervous system development. Further studies confirmed that E2 could induce luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and mRNA expression in prepubertal grass carp pituitary in vivo and in vitro. In the pituitary, LH and FSH regulation by E2 were mediated by both ERβ and GPER1. Apparently, E2-induced LHβ and FSHβ mRNA expression were mediated by adenylyl cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C, and Ca2+/calmodulin/CaM-dependent protein kinase II pathways. In addition to LH and FSH, E2 could also induce growth regulation by estrogen in breast cancer 1 (a novel regulator for pituitary development) mRNA expression in grass carp pituitary cells. These results, as a whole, suggested that E2 could play an important role in gonadotropin hormone release and pituitary development in prepubertal grass carp.

Macrophage colony-stimulating factor (M-CSF) is an intermediate in the process of luteinizing hormone-induced decrease in natriuretic peptide receptor 2 (NPR2) and resumption of oocyte meiosis

BackgroundLuteinizing hormone (LH) regulation of the ligand, natriuretic peptide precursor type C, and its receptor, natriuretic peptide receptor 2 (NPR2), is critical for oocyte maturation; however, the mechanism is not fully understood. Macrophage colony-stimulating factor (M-CSF) has recently been shown to be involved in oocyte maturation and ovulation. In the present study we determined whether or not M-CSF plays a role in the intermediate signal that mediates LH regulation of NPR2 in resumption of oocyte meiosis.MethodsImmature female C57BL/6 mice were injected i.p. with 5 IU of equine chorionic gonadotropin (eCG) to stimulate follicle development. After 44–48 h, the eCG-stimulated mice were injected i.p. with an ovulatory dose of 5 IU of human chorionic gonadotropin (hCG). The ovaries were excised at selected times. Pre-ovulatory follicles (POFs) and cumulus-oocyte complexes were cultured in different media. Immunohistochemical and quantitative real-time PCR analyses were used to assess the expression of M-CSF, M-CSF receptor (M-CSF-R), and NPR2. The presence of germinal vesicle breakdown (GVBD) was examined under a stereomicroscope to morphologically evaluate resumption of oocyte meiosis.ResultsNPR2 was mainly expressed in cumulus cells of pre-ovulatory follicles, while M-CSF and M-CSF-R were expressed in both mural granulosa and cumulus cells. The levels of M-CSF/M-CSF-R and NPR2 decreased within 4 h after treatment of hCG. M-CSF not only reduced the expression of NPR2 mRNA via its receptor (M-CSF-R), but also increased the proportion of GVBD in oocytes.ConclusionM-CSF serves as an intermediate signal, thus inducing a vital decrease in the NPR2 levels in cumulus cells, and regulates the process of LH-induced resumption of meiosis.

Differential expression of microRNAs in luteinising hormone-treated mouse TM3 Leydig cells.

Testosterone is primarily produced by Leydig cells of the mammalian male gonads. The cellular functions of Leydig cells are regulated by the hypothalamus-pituitary-gonad axis, whereas the microRNA (miRNA) changes of LH-treated Leydig cells are unknown. Mouse TM3 Leydig cells were treated with LH, and deep sequencing showed that 29 miRNAs were significantly different between two groups (fold change of >1.5 or <0.5, p<.05), of which 27 were upregulated and two were downregulated. The differential expression of miR-29b-3p, miR-378b, miR-193b and miR-3695 was confirmed by quantitative real-time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs were involved in the Wingless and INT-1, adenosine 5'-monophosphate-activated protein kinase, NF-kappa B and Toll-like receptor signalling pathways. Results showed that miRNAs might be involved in the regulation of LH to Leydig cells.

Architecture of GnRH-Gonadotrope-Vasculature Reveals a Dual Mode of Gonadotropin Regulation in Fish.

The function and components of the hypothalamic-pituitary axis are conserved among vertebrates; however, in fish, a neuroglandular mode of delivery (direct contact between axons and endocrine cells) was considered dominant, whereas in tetrapods hypothalamic signals are relayed to their targets via the hypophysial portal blood system (neurovascular delivery mode). By using a transgenic zebrafish model we studied the functional and anatomical aspects of gonadotrope regulation thus revisiting the existing model. FSH cells were found to be situated close to the vasculature whereas the compact organization of LH cells prevented direct contact of all cells with the circulation. GnRH3 fibers formed multiple boutons upon reaching the pituitary, but most of these structures were located in the neurohypophysis rather than adjacent to gonadotropes. A close association was observed between FSH cells and GnRH3 boutons, but only a fifth of the LH cells were in direct contact with GnRH3 axons, suggesting that FSH cells are more directly regulated than LH cells. GnRH3 fibers closely followed the vasculature in the neurohypophysis and formed numerous boutons along these tracts. These vessels were found to be permeable to relatively large molecules, suggesting the uptake of GnRH3 peptides. Our findings have important implications regarding the differential regulation of LH and FSH and contradict the accepted notion that fish pituitary cells are mostly regulated directly by hypothalamic fibers. Instead, we provide evidence that zebrafish apply a dual mode of gonadotrope regulation by GnRH3 that combines both neuroglandular and neurovascular components.

Plasma insulin-like peptide 3 concentrations are acutely regulated by luteinizing hormone in pubertal Japanese Black beef bulls

Insulin-like peptide 3 (INSL3) is a major secretory product of testicular Leydig cells. The mechanism of acute regulation of INSL3 secretion is still unknown. The present study was undertaken in pubertal beef bulls to (1) determine the temporal relationship of pulsatile secretion among LH, INSL3, and testosterone and (2) monitor acute regulation of INSL3 secretion by LH using GnRH analogue and hCG. Blood samples were collected from Japanese Black beef bulls (N = 6) at 15-minute intervals for 8 hours. Moreover, blood samples were collected at −0.5, 0, 1, 2, 3, 4, 5, and 6 hours after GnRH treatment and −0.5, 0, 2, 4, and 8 hours on the day of treatment (Day 0), and Days 1, 2, 4, 8, and 12 after hCG treatment. Concentrations of LH, INSL3, and testosterone determined by EIAs indicated that secretion in the general circulation was pulsatile. The frequency of LH, INSL3, and testosterone pulses was 4.7 ± 0.9, 3.8 ± 0.2, and 1.0 ± 0.0, respectively, during the 8-hour period. Seventy percent of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse had occurred. The mean increase (peak per basal concentration) of testosterone pulses was higher (P < 0.001) than that of INSL3 pulses. After GnRH treatment, LH concentrations increased (P < 0.01) dramatically 1 hour after treatment and remained high (P < 0.05) until the end of sampling, whereas an elevated (P < 0.05) INSL3 concentration occurred at 1, 2, 5, and 6 hours after treatment. Testosterone concentrations increased (P < 0.01) 1 hour after the treatment and remained high until the end of sampling. After hCG treatment, an increase of INSL3 concentration occurred at 2 and 4 hours, and Days 2, 4, and 8 after treatment (P < 0.05), whereas in case of testosterone, concentrations remained high (P < 0.01) until Day 8 after treatment. The increase (maximum per pretreatment concentration) of INSL3 concentrations after injecting GnRH or hCG was much lower (P < 0.001) than that of testosterone. In conclusion, secretion of INSL3 in blood of bulls occurred in a pulsatile manner. We inferred an acute regulation of INSL3 by LH in bulls because INSL3 concentrations increased immediately after endogenous and exogenous LH stimulation. The increase of INSL3 concentrations by LH was much lower than that of testosterone in bulls.

Novel pituitary actions of TAC3 gene products in fish model: receptor specificity and signal transduction for prolactin and somatolactin α regulation by neurokinin B (NKB) and NKB-related peptide in carp pituitary cells.

TAC3 is a member of tachykinins, and its gene product neurokinin B (NKB) has recently emerged as a key regulator for LH through modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also a novel tachykinin-like peptide called NKB-related peptide (NKBRP), and the pituitary actions of these TAC3 gene products are still unknown. Using grass carp as a model, the direct effects and postreceptor signaling for the 2 TAC3 products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP similar to that of other fish species. In carp pituitary cells, NKB and NKBRP treatment did not affect LH release and gene expression but up-regulated prolactin (PRL) and somatolactin (SL)α secretion, protein production, and transcript expression. The stimulation by these 2 TAC3 gene products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by adenylate cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C and Ca(2+)/calmodulin/Ca(2+)/calmodulin-dependent protein kinase II activation. The signal transduction for the corresponding responses on PRL release and mRNA expression were also similar, except that the protein kinase C component was not involved. These findings suggest that the 2 TAC3 gene products do not play a role in LH regulation at the pituitary level in carp species but may serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping postreceptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.

The effect of the intracervical application of follicle-stimulating hormone or luteinizing hormone on the pattern of expression of gonadotrophin receptors in the cervix of non-pregnant ewes.

During the periovulatory period, the cervix relaxes in response to changes in circulating concentrations of reproductive hormones. The present study investigated the role of gonadotrophins in cervical function by examining the expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) and their mRNAs following intracervical treatment with either FSH or LH. Eighteen ewes were assigned to four groups, and they were then treated with progestagen sponges and PMSG to synchronize their oestrous cycles. Intracervical treatments were given 24h after sponge removal as follows: Group 1: FSH 2mg; Group 2: LH 2mg; Group 3: Vehicle and Group 4: Control. Cervices were collected 54h after sponge removal and then divided into three regions. The expression of FSHR and LHR was determined by immunohistochemistry and FSHR mRNA and LH mRNA by in situ hybridization. The expression of LHR, FSHR and their respective mRNAs was compared in six tissue layers (luminal epithelium, subepithelial stroma, circular, longitudinal and transverse muscle and serosa) and in three cervical regions (vaginal, mid and uterine). The results showed that FSH increased transcription of the FSHR gene and the levels of its receptor, but only in subepithelial stroma of the cervix. FSH also increased the levels of LHR in the cervix, but only in the muscle layers. LH had no effect on the levels of FSHR despite the fact that it did increase the level of transcription of the FSHR gene and LH also increased the levels of its own receptor in the cervix, but only in the muscle layers, and this action was independent of increased levels of transcription of the LHR gene. These findings suggest multiple levels of regulation of cervical LH and FSH receptors and that the gonadotrophins may have a role in relaxation of the cervix during oestrus by regulating their own receptors.

PI3K signalling in GnRH actions on dispersed goldfish pituitary cells: Relationship with PKC-mediated LH and GH release and regulation of long-term effects on secretion and total cellular hormone availability

Goldfish pituitary cells are exposed to two GnRHs, salmon (s)GnRH and chicken (c)GnRH-II. Phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) both participate in acute sGnRH- and cGnRH-II-stimulated LH and GH release. Using goldfish pituitary cells, we examined the relationship between PI3K and PKC in acute LH and GH secretion, and PI3K involvement in chronic hormone release and total LH and GH availability. The PI3K inhibitor LY294002 did not affect PKC agonists-induced LH or GH release, and PKC agonists did not alter PI3K p85 phosphorylation, suggesting PKC activation is not upstream of PI3K in acute hormone release. In 2, 6, 12 and 24h treatments, LY294002 did not affect LH release but stimulated total LH availability at 6h. sGnRH stimulatory actions on LH release and total availability at 12 and 24h, and cGnRH-II effects on these parameters at 6h were inhibited by LY294002. LY294002 enhanced basal GH release at 2 and 6h, but reduced total GH at 12 and 24h. Increased GH release was seen following 6, 12 and 24h of sGnRH, and 2, 6 and 24h of cGnRH-II treatment but total GH availability was only elevated by 24h cGnRH-II treatment. Whereas LY294002 inhibited GH release responses to sGnRH at 12h and cGnRH-II at 6h, it attenuated cGnRH-II-elicited, but not sGnRH-induced, effects on total GH. These results indicate that PI3K differentially modulates long-term basal and GnRH-stimulated hormone release, and total hormone availability, in a time-, cell-type-, and GnRH isoform-selective manner.

Somatostatin Receptors Type 2 and 5 Expression and Localization During Human Pituitary Development

Somatostatin (SRIF), by acting mainly through sst2 and sst5 receptors, is a potent inhibitor of hormonal secretion by the human anterior pituitary gland. However, the pattern of protein expression of these SRIF receptors remains unknown during pituitary development. To get further insights into the physiological role of SRIF receptors in human development and pituitary function, the present study examined the developmental expression of the sst2 and sst5 receptors in the individual cell types of the anterior human pituitary. Thirteen fetal human pituitaries were investigated between 13 to 38 weeks of gestation (WG) by double-labeling immunofluorescence with antibodies raised against sst2 or sst5 receptors and GH, LH, FSH, TSH, or pro-opiomelanocortin proteins. SRIF immunoreactivity in the hypothalamus and median eminence was investigated at the same developmental ages. Immunoreactivity for the sst2 receptor was evident as early as 13 to 15 WG and onward mainly in TSH-, LH-, and FSH-expressing cells, whereas sst5 immunoreactivity was apparent at the late development stages (35-38 WG). GH-expressing cells mainly expressed sst5 immunoreactivity. SRIF-positive fibers and cells were detected as soon as 13 to 16 WG in the hypothalamus and median eminence and their densities increased with gestational age. The early appearance of hypothalamic SRIF cells and fibers suggests a physiological link between SRIF and its receptors during pituitary development. Whereas sst2 receptors might play a primary role in the differentiation and regulation of TSH, LH, and FSH cells, sst5 receptors appear to be mainly involved in GH regulation from birth onward.