GnRH Synthesis Research Articles

6544 M6A methylation of PLCβ in the hypothalamus regulates GnRH synthesis and secretion by interfering Ca2+ signaling pathway

Abstract Disclosure: X. Yin: None. S. Zang: None. P. Li: None. Backgrounds: In recent years, the incidence of precocious puberty has increased rapidly, posing a serious threat to girls' physical and mental health. Epigenetic changes, especially m6A methylation modification, may be an important influencing factor. Researches had found that m6A modification plays an irreplaceable role in the development and functional homeostasis of the nervous system. A variety of factors could changed RNA m6A level in the hypothalamus and affect the process of adolescent sexual development. In the study,we aimed to explore the specific role of m6A methylation in precocious puberty, and to identify its downstream target genes by multi-omics approach and to verify its function. Hypothesis The demethylase FTO regulated the m6A modification of the target gene in the hypothalamus to affect the synthesis and secretion of GnRH and regulate the initiation of puberty. Methods: The modification abundance of m6A was investigated by the girls with central precious puberty (CPP). The changes of m6A modification in total RNA and the expression of important modulator genes were detected by colorimetry and qRT-PCR respectively in the hypothalamic ARC nucleus of female rats at three different developmental stages (juvenile, early puberty and puberty) and precocious rat model. MeRIP and mRNA sequencing was used to screen target genes which related to puberty development. Knockdown or overexpression of FTO were constructed using plasmid or lentivirus transfection. The target mRNA targeted regulated by FTO was verified with RNA-seq, qRT-PCR, WB and Flow cytometry. Results This study confirmed that the level of RNA m6A in the peripheral blood of the precocious group was significantly higher than that of the control group. The level of RNA m6A in the hypothalamus of female rats gradually increases with the development of puberty, and the expression of demethylase gradually decreases. MeRIP-seq combined with mrNA-seq screening analysis showed that the m6A modification of PLCβ in the ARC nucleus of the hypothalamus was reduced, and its mRNA expression was significantly increased in sexually precocious female rats. Combined with bioinformatics analysis, it was found that there were multiple m6A modified motif sites on the mRNA of PLCβ, and luciferase experiment confirmed that there was a regulatory relationship between them. After over-expressing FTO, the mRNA and protein expression of PLCβ were significantly increased, and the up-regulation of FTO expression induced a significant increase in intracellular Ca2+ concentration, the expression of CAM, and the activation of Ca2+ signaling pathway. Conclusions In the ARC nucleus of the hypothalamus, FTO affects the synthesis and secretion of GnRH by regulating the m6A modification level of PLCβ through the Ca2+ signaling pathway, and interferes with the initiation of female sexual development. Presentation: 6/1/2024

Mechanism of LH release after peripheral administration of kisspeptin in cattle.

Kisspeptin modulates GnRH secretion in mammals and peripheral administration of 10-amino acid fragment of kisspeptin (Kp10) induces LH release and ovulation in cattle. Experiments were done to determine if iv administration of kisspeptin will activate GnRH neurons (i.e., after crossing the blood-brain barrier) and if pre-treatment with a GnRH receptor blocker will alter kisspeptin-induced LH release (from gonadotrophs) and ovulation. In Experiment 1, cows (n = 3 per group) were given human-Kisspeptin10 (hKp10; 3 x 15 mg iv at 60-min intervals) or normal saline and euthanized 150 min after treatment was initiated. Every 20th free-floating section (50 μm thickness) from the preoptic area to hypothalamus was double immunostained to colocalize GnRH- (DAB) and activated neurons (cFOS; Nickel-DAB). Kisspeptin induced plasma LH release from 15 to 150 min (P = 0.01) but the proportion of activated GnRH neurons did not differ between groups (5.8% and 3.5%, respectively; P = 0.11). Immunogold electron microscopy detected close contacts between kisspeptin fibers and GnRH terminals in the median eminence. In Experiment 2, pubertal heifers (n = 5 per group) were treated with 1) hKp10 iv, 2) Cetrorelix (GnRH antagonist; im) + hKp10 iv or 3) saline on Day 6 of the follicular wave under low-progesterone condition. A rise in plasma LH concentration was detected from 15 to 240 min in the hKp10 group but not in cetrorelix or control group (P<0.001). Ovulations were detected only in the hKp10 group (4/5; P = 0.02). Cetrorelix treatment was associated with regression of the preovulatory dominant follicle and emergence of a new follicular wave 3.4±0.75 days after the treatment in all five heifers. Results support the hypothesis that the effect of peripheral kisspeptin is mediated downstream of GnRH synthesis and does not involve GnRH-independent LH release from gonadotrophs. Peripheral kisspeptin may release pre-synthesized GnRH from the nerve terminals in areas outside the blood-brain barrier.

Study of estrogen, progesterone and glucose level in cows during overt and silent estrus

Significant economic losses result from increased days open because of failure to detect estrus. Studies (Callahan, et al., 1971; Kesler,et al., 1977) suggest that the endocrine balances required to support normal estrus cycles and for the reestablishment of fertility are restored gradually after calving. Silent estrus, or the lack of behavioral estrus expression, is a common occurrence (50 to 80%) at the first postpartum ovulation. However, the incidence of silent estrus decreases in the second and third estrous cycles (Bulman,1980 ; Labhsetwar, et al., 1946; Morrow, et al., 1966). Expression of estrus in cattle is dependent on sufficient secretion of endogenous E2 to provoke the standing response (Carrick and Shelton. 1969) Estrogen plays a key role in the regulation of endocrine and behavioral events associated with the estrus cycle. E2 plays a pivotal role in the induction of estrus behavior and ovulation (Pfaff, 2005). In many experiments that are performed to study the female reproduction, estrus is artificially induced by administering E2 (Fabre-Nys et al., 1993). Estrogen plays a central role in triggering the gonadotropin surge and ovulation as well as in facilitating the estrus behavior, and thus estrogen indirectly synchronizes mating and ovulation. The patterns of GnRH synthesis and pulsatile release from the hypothalamus are mainly regulated by E2 (Smith and Jennes, 2001) and progesterone (Richer et al., 2005 ;Zalanyi, 2001). E2 stimulates LH synthesis, but at levels below a certain threshold value it inhibits the release of LH. Above this threshold, the inhibitory effect on LH release switches to stimulatory effect (Reinecke and Deuflhard, 2007), which results in the LH surge. Progesterone plays an essential role in various reproductive functions, including regulating the length of the estrous cycle, maintaining pregnancy. Progesterone is high during the luteal phase and in pregnancy, however the levels decline if the animal fails to conceive. Progesterone has been implicated in inhibiting estrogen secretion and ovulation (Echternkamp et al. 1973 and Henricks, 1971). Blood glucose appear to be one of the key nutrients affecting cyclicity in farm animals and a minimum level of 40-60 mg/ml is required to maintain the physiological processes of the body (Duke, 1970). According to Dowine and Gelman (1976), low blood glucose may be associated with infertility. Estrus is negatively affected by alterations in blood metabolites and hormone profiles including glucose, insulin, IGF-I, non-esterified fatty acids, β-hydroxybutyrate (Wathes et al., 2007). Nadiu and Rao

Irisin in the primate hypothalamus and its effect on GnRH in vitro.

Irisin, encoded by the FNDC5 gene, is a recently discovered endocrine factor mainly secreted as a myokine and adipokine. However, irisin/FNDC5 expression has also been reported in different other organs including components of the reproductive axis. Yet, there is the scarcity of data on FNDC5/irisin expression, regulation and its reproductive effects, particularly in primates. Here, we report the expression of FNDC5/irisin, along with PGC1A (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and ERRA (estrogen-related receptor alpha), in components of the reproductive axis of marmoset monkeys. Hypothalamic FNDC5 and ERRA transcript levels are developmentally regulated in both male and female. We further uncovered sex-specific differences in FNDC5, ERRA and PGC1A expression in muscle and the reproductive axis. Moreover, irisin and ERRα co-localize in the marmoset hypothalamus. Additionally, in the arcuate nucleus of rhesus monkeys, the number of irisin+ cells was significantly increased in short-term fasted monkeys as compared to ad libitum-fed monkeys. More importantly, we observed putative interaction of irisin-immunoreactive fibers and few GnRH-immunoreactive cell bodies in the mediobasal hypothalamus of the rhesus monkeys. Functionally, we noted a stimulatory effect of irisin on GnRH synthesis and release in mouse hypothalamic neuronal GT1-7 cells. In summary, our findings show that FNDC5 and irisin are developmentally, metabolic-status dependently and sex-specifically expressed in the primate hypothalamic-pituitary-gonadal axis and exert a stimulatory effect on GnRH expression and release in mouse hypothalamic cells. Further studies are required to confirm the reproductive effects of irisin in vivo and to illuminate the mechanisms of its regulation.

Gonadoliberin – Synthesis, Secretion, Molecular Mechanisms and Targets of Action

Decapeptide gonadoliberin (GnRH) is the most important regulator of the hypothalamic-pituitary-gonadal (HPG) axis that controls the synthesis and secretion of the luteinizing and follicle-stimulating hormones by gonadotrophs in the adenohypophysis. GnRH is produced by the specialized hypothalamic neurons using the site-specific proteolysis of the precursor protein and is secreted into the portal pituitary system, where it binds to the specific receptors. These receptors belong to the family of G protein-coupled receptors, and they are located on the surface of gonadotrophs and mediate the regulatory effects of GnRH on the gonadotropins production. The result of GnRH binding to them is the activation of phospholipase C and the calcium-dependent pathways, the stimulation of different forms of mitogen-activated protein kinases, as well as the activation of the enzyme adenylyl cyclase and the triggering of cAMP-dependent signaling pathways in the gonadotrophs. The gonadotropins, kisspeptin, sex steroid hormones, insulin, melatonin and a number of transcription factors have an important role in the regulation of GnRH1 gene expression, which encodes the GnRH precursor, as well as the synthesis and secretion of GnRH. The functional activity of GnRH-producing neurons depends on their migration to the hypothalamic region at the early stages of ontogenesis, which is controlled by anosmin, ephrins, and lactosamine-rich surface glycoconjugate. Dysregulation of the migration of GnRH-producing neurons and the impaired production and secretion of GnRH, lead to hypogonadotropic hypogonadism and other dysfunctions of the reproductive system. This review is devoted to the current state of the problem of regulating the synthesis and secretion of GnRH, the mechanisms of migration of hypothalamic GnRH-producing neurons at the early stages of brain development, the functional activity of the GnRH-producing neurons in the adult hypothalamus and the molecular mechanisms of GnRH action on the pituitary gonadotrophs. New experimental data are analyzed, which significantly change the current understanding of the functioning of GnRH-producing neurons and the secretion of GnRH, which is very important for the development of effective approaches for correcting the functions of the HPG axis.

Negative feedback regulation of the secretion and actions of GnRH in male ruminants

The roles of testicular hormones in the negative feedback regulation of the secretion and actions of GnRH in male domestic ruminants are reviewed, concentrating mainly on research conducted with rams. Testicular steroids have major feedback actions directly at the hypothalamus to inhibit the secretion of GnRH, although it is apparent that, under certain circumstances, the steroids also have actions directly at the pituitary gland. Further research is necessary to delineate these actions and to determine the contribution of testosterone and its primary metabolites, dihydrotestosterone and oestradiol, to negative feedback on the hypothalamo-pituitary unit. Since GnRH neurones do not possess receptors for steroids, testicular steroids must evoke other neuronal pathways to influence GnRH producing neurones. While the opioids may be important in this regard, it is necessary to determine which other neuronal pathways may also be involved to understand fully the mechanism of action of testicular steroids. It appears that the feedback regulation of the secretion of LH can be accounted for by testicular steroids, whereas the secretion of FSH is influenced by inhibin and steroids, and possibly the recently isolated proteins follistatin and activin. The actions of inhibin to suppress the secretion of FSH occur at the pituitary gland and not on the synthesis or secretion of GnRH. There is a complex interaction between testosterone and inhibin in the control of FSH secretion that results in synergistic effects during the non-breeding season but not during the breeding season. Activin has been shown to have FSH-stimulating properties and follistatin has been shown to have FSH-inhibiting properties, but it is unknown if these proteins play a physiological role in the feedback regulation of FSH in male domestic ruminants.

Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish.

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey.

Effects of active immunization against GnRH versus surgical castration on hypothalamic-pituitary function in boars

The objective was to compare effects of anti-GnRH immunization (immunocastration) versus surgical castration on hypothalamic-pituitary function in boars. Thirty-six boars were randomly divided into three groups (n = 12/group): control, surgically castrated, or immunized against GnRH at 10 wk of age (boostered 8 wk later). Compared to intact boars, immunocastration reduced (P < 0.05) serum concentrations of LH, FSH, testosterone and inhibin B and caused severe testicular atrophy, whereas surgical castration increased (P < 0.05) serum concentrations of LH and FSH. Both immunocastration and surgical castration consistently reduced hypothalamic GnRH synthesis, with decreased (P < 0.05) mRNA expressions of GnRH, GnRH up-stream gatekeeper genes kiss1 and its receptor (GPR54), and androgen receptor in the hypothalamic arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV), as well as GnRH content in the median eminence. Inconsistently, mRNA expressions of gonadotropin-inhibitory hormone (GnIH) in ARC and AVPV as well as its receptor (GPR147) in pituitary were selectively reduced (P < 0.05), but mRNA expressions of estrogen receptor alpha and aromatase (CPY17A1) in pituitary were selectively increased (P < 0.05) in surgical castrates. In response to selectively attenuated suppressive signaling from GnIH and testosterone, mRNA expressions of GnRH receptor (GnRHR), LH-β and FSH-β in pituitary were increased (P < 0.05) in surgical castrates, whereas these pituitary gene expressions were decreased (P < 0.05) in immunocastrates, due to loss of hypothalamic GnRH signaling. We concluded that immunocastration and surgical castration consistently reduced hypothalamic GnRH synthesis due to a testosterone deficiency disrupting testosterone-Kisspeptin-GPR54-GnRH signaling pathways. Furthermore, selectively attenuated GnIH and testosterone signaling in the pituitary increased gonadotropin production in surgical castrates.

Physiological interactions between the hypothalamic-pituitary-gonadal axis and spleen in rams actively immunized against GnRH

Hypothalamic-pituitary-gonadal (HPG) axis is strongly implicated in the regulation of immune system. The objective was to determine the effects of immunocastration on splenic reproduction- and immunity-related gene expressions, and serum cytokine profiles in rams. Forty rams were randomly allocated into three groups: control (n=14); surgically castrated (n=13); or immunized (n=13) against 100μg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 6months of age (with a booster 2months later). Blood samples (for hormone and immune cytokine profiles) were collected at 1-month intervals until rams were slaughtered (10months). Compared to intact controls, anti-GnRH immunization reduced (P<0.05) serum concentrations of LH, FSH, and testosterone. Reduced testosterone abrogated its inhibitor feedback effect on the synthesis of GnRH in spleen, as evidenced by increased (P<0.05) protein content and mRNA expressions of GnRH, and simultaneously decreased (P<0.05) mRNA expressions of androgen receptor in spleen. In parallel with the increased GnRH production in spleen, the mRNA expressions of interleukin (IL)-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α) as well as lymphocyte marker CD4, CD8 and CD19 molecules were increased (P<0.05) in spleen. Consistently, serum concentrations of IL-2, IL-4, IL-6, TNF-α were increased (P<0.05) in rams following immunization. Similarly, deprivation of testosterone by surgical castration also increased (P<0.05) GnRH and thus immune cytokine expressions in spleen. Collectively, our data suggested that immunocastration increased GnRH production in spleen by abrogating the inhibitory feedback effects from testosterone, consequently improving the immune markers of spleen and serum immune cytokines in rams.

Effects of Huang Bai (Phellodendri Cortex) and Three Other Herbs on GnRH and GH Levels in GT1-7 and GH3 Cells.

The present study was to evaluate the effects of Huang Bai, Zhi Mu, Mai Ya, and Xia Ku Cao on hormone using the GT1–7 and GH3 cells. The GT1–7 and GH3 cell lines were incubated with DW; DMSO; and 30, 100, or 300 μg/mL of one of the four extract solutions in serum-free media for 24 hours. The MTT assay was performed to determine the cytotoxicity of the four herbs. The GT1–7 and GH3 cells were incubated in DW, estradiol (GT1–7 only), or noncytotoxic herb solutions in serum-free medium for 24 hours. A quantitative RT-PCR and western blot were performed to measure the GnRH expression in GT1–7 cells and GH expression in GH3 cells. Huang Bai, Zhi Mu, Xia Ku Cao, and Mai Ya inhibited the GnRH mRNA expression in GT1–7 cells, whereas Huang Bai enhanced GH mRNA expression in GH3 cells. Additionally, Xia Ku Cao inhibited GnRH protein expression in GT1–7 cells and Huang Bai promoted GH protein expression in GH3 cells. The findings suggest that Huang Bai can delay puberty by inhibiting GnRH synthesis in the hypothalamus while also accelerating growth by promoting GH synthesis and secretion in the pituitary.

The negative effects of chronic exposure to isoflurane on spermatogenesis via breaking the hypothalamus-pituitary-gonadal equilibrium

This study aims to investigate the negative effects of chronic exposure to isoflurane on spermatogenesis and explore the underlying mechanisms. Sixty male rats were randomly allocated to two groups: control group, receiving no treatment, and anesthesia group, administrated exposure to isoflurane (2 ppm) for 25 consecutive days (1 h/day). The negative effects of chronic exposure to isoflurane were evaluated by analyzing the median eminence GnRH content, the relevant hormone levels, some sperm parameters and the mRNA expressions for some reproduction-related genes. Isoflurane significantly decreased the GnRH content and the serum gonadotrophin levels compared with the control group (p < 0.01). Meanwhile, the mRNA expressions of GnRH in hypothalamus, GnRH receptor, luteinizing hormone (LH)-β and follicle-stimulating hormone (FSH)-β in pituitary, and LH receptor and FSH receptor in testes were also significantly inhibited (p < 0.01). Furthermore, the mRNA expressions of androgen receptor (AR), kisspeptin encoded gene (Kiss-1) and its receptor (GPR54) in hypothalamus were significantly diminished by isoflurane (p < 0.01). The results indicated that chronic exposure to isoflurane diminished the synthesis and secretion of GnRH by inhibiting the androgen-AR-Kisspeptin-GPR54 pathway and breaking the hypothalamic-pituitary-gonadal equilibrium, and therefore it could inhibit spermatogenesis.

Molecular identification of GnIH/GnIHR signal and its reproductive function in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)

Gonadotropin-inhibitory hormone (GnIH) and its receptor (GnIHR) play an important role in reproduction regulation in birds, mammals and some teleost species. In protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides), the GnIH/GnIHR signaling pathway and its reproductive function have not been addressed yet. In this study, GnIH and GnIHR in orange-spotted grouper were characterized. gGnIH possessed three putative peptides (gGnIH-I, -II, -III), while gGnIHR showed the characteristics of G protein-coupled receptor and was clustered with GPR147. Functional assays demonstrated that three synthetic gGnIH peptides significantly decreased the forskolin-induced CRE promoter activity, but only gGnIH-I could significantly decrease SRE promoter activity in COS-7 cells transfected with gGnIHR. During the process of ovarian differentiation and development, gGnIH mRNA level in hypothalamus was low at the gonadal primordium stage with gonia, then increased significantly at the early differentiated gonad with primary growth oocytes, while decreased significantly at the developing gonads with cortical-alveolus and vitellogenic stage oocytes. During MT-induced sex reversal, gGnIH mRNA level in hypothalamus increased significantly when the fish completely reversed from female to male. However, gGnIHR mRNA level in pituitary decreased significantly in intersex and completely reversed male fish. Intraperitoneal injection (i.p.) of three gGnIH peptides significantly decreased GnRH1 mRNA levels in hypothalamus, and gGnIH-II significantly inhibited synthesis of LHβ in pituitary. In summary, we firstly identified the GnIH/GnIHR signal in protogynous orange-spotted grouper, which might be involved in the regulation of the reproductive function of sex differentiation, gonadal development and sex reversal via regulating the synthesis of both GnRH and GtH.

Effects of Caponization on Expression of Gonadotropin-Releasing Hormone-I and Gonadotropin Subunits Genes in Roosters.

We evaluated the effects of caponization on mRNA levels of gonadotropin-releasing hormone-I (GnRH-I), gonadotropin subunit and other hypothalamic and hypophyseal peptide genes in male chicken. Thirty roosters (25 d) with similar weight were equally divided into the experimental (capons) and control (sham-operated males) groups randomly. Caponization was performed at 28 days of age and birds were slaughtered at 140 days of age. Caponization resulted in increasing levels of luteinizing hormone β (LHβ) and follicle-stimulating hormone β (FSHβ) mRNA in the pituitary gland and levels of LH and FSH in serum (P<0.05 or P<0.01). There were no significant differences in levels of GnRH-I, Gonadotropin releasing hormone receptor (GnRHR), neuropeptide Y (NPY) and Proopiomelanocortin (POMC) mRNA between the two groups. Capons exhibited lower levels of follistatin (FS), estrogen receptor α (ERα) and higher levels of androgen receptor (AR) mRNA in the pituitary gland compared with sham-operated males (P<0.05). These results suggest that increased LH and FSH concentrations in serum and LHβ and FSHβ mRNA levels in pituitary after castration are not depended on GnRH synthesis. And changed expression of ERα, AR and FS genes in the pituitary gland may be important components of regulating gonadotropin in capons.

Exogenous kisspeptin administration as a probe of GnRH neuronal function in patients with idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) results from defective synthesis, secretion, or action of GnRH. Kisspeptin is a potent stimulus for GnRH secretion. We probed the functional capacity of the GnRH neuronal network in patients with IHH. Eleven subjects with congenital IHH (9 men and 2 women) and one male subject who underwent reversal of IHH were studied. Six of the twelve subjects had an identified genetic cause of their IHH: KAL1 (n = 1), FGFR1 (n = 3), PROKR2 (n = 1), GNRHR (n = 1). Subjects underwent q10 min blood sampling to measure GnRH-induced LH secretion at baseline and in response to intravenous boluses of kisspeptin (0.24 nmol/kg) and GnRH (75 ng/kg) both pre- and post-six days of treatment with exogenous GnRH (25 ng/kg sc every 2 h). All subjects with abiding IHH failed to demonstrate a GnRH-induced LH response to exogenous kisspeptin. In contrast, the subject who achieved reversal of his hypogonadotropism demonstrated a robust response to kisspeptin. The functional capacity of the GnRH neuronal network in IHH patients is impaired, as evidenced by their inability to respond to the same dose of kisspeptin that effects a robust GnRH-induced LH response in healthy men and luteal-phase women. This impairment is observed across a range of genotypes, suggesting that it reflects a fundamental property of GnRH neuronal networks that have not been properly engaged during pubertal development. In contrast, a patient who had experienced reversal of his hypogonadotropism responded to exogenous kisspeptin.

Glutamate supply positively affects serum cholesterol concentrations without increases in total protein and urea around the onset of puberty in goats

Different neurotransmitter and neuromodulatory systems regulate synthesis and secretion of GnRH. Whereas the endocrine and neural systems are activated in response to the metabolic status and the circulating levels of specific blood metabolites, glutamate receptors have been reported at hepatic level. This study evaluated the possible effect of glutamate supplementation upon changes in serum concentrations across time for total protein (TP), urea (UR) and cholesterol (CL) around the onset of puberty in goats. Prepuberal female goats (n=18) were randomly assigned to: (1) excitatory amino acids group, GLUT, n=10; 16.52±1.04kg live weight (LW), 3.4±0.12 body condition score (BCS) receiving an i.v. infusion of 7mgkg−1 LW of l-glutamate, and (2) Control group, CONT, n=8; 16.1±1.04kg LW, 3.1±0.12 BCS. General averages for LW (23.2±0.72kg), BCS (3.37±0.10 units), serum TP (65.28±2.46mgdL−1), UR (23.42±0.95mgdL−1), CL (77.89±1.10mgdL−1) as well as the serum levels for TP and UR across time did not differ (P>0.05) between treatments. However, while GLUT positively affected (P<0.05) both the onset (207±9 vs. 225±12 d) and the percentage (70 vs. 25%) of females showing puberty, a treatment×time interaction effect (P<0.05) was observed in the GLUT group, with increases in serum cholesterol, coincident with the onset of puberty. Therefore, in peripuberal glutamate supplemented goats, serum cholesterol profile could act as a metabolic modulator for the establishment of puberty, denoting also a potential role of glutamate as modulator of lipid metabolism.

Active immunisation against pregnenolone reduces testicular steroidogenesis and GnRH synthesis in rabbits

To investigate the effects of active immunisation against pregnenolone on reproductive traits in rabbits, 16 early pubertal male rabbits (4mo old) were randomly and equally allocated into two groups, control or immunised against pregnenolone–hemisuccinate–BSA in Freund's adjuvant (with a booster 4wk later). Blood samples (for antibody titres and hormone concentrations) were collected at 2 or 4wk-intervals after immunisation until rabbits were killed, 24wk after the primary immunisation. Compared to controls, rabbits immunised against pregnenolone had increased serum antibody titres (P<0.01) and decreased serum concentrations of both testosterone and LH (P<0.01 for each). At 24wk after the primary immunisation, testes were severely atrophied, spermatogenesis was arrested and steroidogenesis was suppressed, as evidenced by lesser amounts of testicular cholesterol side-chain cleavage cytochrome P-450 and 17α-hydroxylase cytochrome P-450 mRNA (P<0.05). Furthermore, the amounts of mRNA for GnRH in the arcuate nucleus (Arc), of the hypothalamus and GnRH receptor and LH-β in the pituitary and genes in sex-hormone negative feedback loops (androgen receptor, oestrogen alpha receptor, kisspeptin encoded gene and kisspeptin receptor) in the Arc were decreased in pregnenolone-immunised rabbits compared to controls (P<0.05). It was concluded that immunisation against pregnenolone directly blocked testicular steroidogenesis, which reduced synthesis of hypothalamic GnRH and subsequently synthesis of pituitary LH by abolishing the permissive action of sex steroids on hypothalamic GnRH neurons, thereby disrupting spermatogenesis. This was apparently the first report that active immunisation against pregnenolone was a means of immunological castration

Active immunization against GnRH reduces the synthesis of GnRH in male rats

We sought to determine the effects of active anti-GnRH immunization on GnRH synthesis in the hypothalamus. Adult male rats (n = 36) were randomly and equally allocated into three groups: Control (no treatment), surgically castrated, or immunized against 50 μg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 12 week of age (with a booster 8 week later). Blood samples (for antibody titers and hormone concentrations) were collected at 2-week intervals until rats were killed (20 week). Compared with intact controls, immunocastration reduced (P < 0.05) serum concentrations of testosterone, LH, and FSH, and GnRH content in the median eminence, reduced the weight of the hypohysis (P < 0.01), and induced testicular atrophy (suppression of spermatogenesis). Furthermore, mRNA expression of GnRH in the hypothalamus, GnRH receptor, LH-β and FSH-β in the pituitary, LH receptor and FSH receptor in the testes, and genes in sex steroid feedback loops (androgen receptor [AR], kisspeptin encoded gene (Kiss-1), and kisspeptin receptor (GPR54) in the hypothalamus were decreased in immunocastrated rats compared with intact controls (P < 0.05). Similarly, surgical castration reduced GnRH in the median eminence as well as mRNA expression of GnRH, AR, Kiss-1, and GPR54 in the hypothalamus (P < 0.05). We concluded that anti-GnRH immunization in adult rats reduced synthesis of hypothalamic GnRH by decreasing androgen–AR–Kisspeptin–GPR54 signaling pathways, and caused dysfunction of the pituitary–testicular axis, thereby suppressing spermatogenesis, resulting in testicular atrophy.

Clinical Genetic Testing for Kallmann Syndrome

During the past 20 years, remarkable advances have been made regarding the molecular basis of many disorders affecting reproduction. This is particularly true for patients who present with delayed puberty due to GnRH deficiency. These affected males and females manifest low serum levels of sex steroids, low or normal gonadotropins, and no other pituitary pathology. The pathophysiology of GnRH deficiency is complex, but 2 major phenotypes are observed. When hypothalamic GnRH gene regulation or GnRH synthesis, secretion, or signaling is impaired, the resulting phenotype is normosmic hypogonadotropic hypogonadism (nHH). However, it is also known that GnRH neurons originate outside of the brain and migrate along with olfactory neurons from the nasal region into the hypothalamus. If this migratory pathway of GnRH and olfactory neurons is disrupted, anosmia accompanies hypogonadotropic hypogonadism, known as Kallmann syndrome (KS).

Candidate mechanisms underlying atypical progesterone profiles as deduced from parameter perturbations in a mathematical model of the bovine estrous cycle

The complex interplay of physiological factors that underlies fertility in dairy cows was investigated using a mechanistic mathematical model of the dynamics of the bovine estrous cycle. The model simulates the processes of follicle and corpus luteum development and its relations with key hormones that interact to control these processes. Several factors may perturb the regular oscillatory behavior of a normal estrous cycle, and such perturbations are likely the effect of simultaneous changes in multiple parameters. The objective of this paper was to investigate how multiple parameter perturbation changes the behavior of the estrous cycle model, so as to identify biological mechanisms that could play a role in the development of cystic ovaries. Cystic ovaries are a common reason for reproductive failure in dairy cows, but much about the causes of this disorder remains unknown. We investigated in which region of the parameter space the model predicts a normal cycle, and when a progesterone pattern occurred with delayed ovulation (indicating a cystic follicle) or delayed luteolysis (indicating a persistent corpus luteum). Perturbation of the initial values for all parameters simultaneously showed 2 specific parameter configurations leading to delayed ovulation or delayed luteolysis immediately. The most important parameter changes in these 2 configurations involve the regulation of corpus luteum functioning, luteolytic signals, and GnRH synthesis, suggesting that these mechanisms are likely involved in the development of cystic ovaries. In the multidimensional parameter space, areas exist in which the parameter configurations resulted in normal cycles. These areas may be separated by areas in which irregular cycle patterns occurred. These irregular patterns thus mark the transition from one stable (normal) situation to another. Interestingly, within a series, there were some cycles with delayed ovulation and some with delayed luteolysis in these patterns. This could represent a situation of resumption of normal cyclicity (e.g., after parturition). In conclusion, the method of parameter perturbation used in the present study is an effective tool to find parameter configurations that lead to progesterone profiles associated with delayed ovulation and delayed luteolysis. Thereby, the model helps to generate hypotheses regarding the underlying cause of the development of cystic ovaries, which could be investigated in future experiments.

Research progress in molecular mechanism of animal seasonal reproduction

Animal seasonal reproduction involves complicated neuroendocrine processes of the hypothalamo-pituitary-gonadal axis. It is dominantly regulated by photoperiod, a crucial environmental cue. Melatonin, as internal photoperiod signal, regulates seasonal reproduction of animals. In recent years, it has been found that Kiss1/GPR54 system, which may influence GnRH secretion evidently, is regulated by both melatonin and feedback action of gonadal steroid hormones. Consequently, Kiss1/GPR54 system may play a key role in seasonal reproduction. Additionally, there exists another potential retrograde control pathway of seasonal breeding, which involves TSH-DIO2/DIO3 system. TSH-DIO2/ DIO3 system affects synthesis and secretion of GnRH and is regulated by melatonin, as well as Kiss1/GPR54 system. In this article, melatonin signal, especially the research advances of Kissl/GPR54 system and TSH-DIO2/DIO3 system were reviewed.