Polyamines: An Emerging Regulator of Gonadotropin Releasing Hormone (GnRH)

INTRODUCTION

Gonadotropin-releasing hormone (GnRH) is a critical neuropeptide that regulates reproductive function by modulating the secretion of gonadotropins from the anterior pituitary. While its regulation is linked through classical pathways involving the steroids, neurotransmitters, and metabolic signals, recent studies highlight the involvement of polyamines as novel regulators of GnRH.^[1–4]

GnRH STRUCTURE AND FUNCTIONS

GnRH, a decapeptide, has a highly conserved 10 amino acid sequence across various vertebrates and has also been identified in invertebrates, highlighting its evolutionary significance. Multiple isoforms of GnRH exist, with at least 50% sequence similarity.^[5,6] Uniquely, GnRH neurones originate from the olfactory placode, migrate to the brain during development, and primarily reside in the ventral forebrain, hypothalamus, preoptic region, and pituitary gland.

The hypophysiotropic GnRH, responsible for stimulating gonadotropin release, exists in several isoforms: GnRH-I, GnRH-II and GnRH-III, with only GnRH-I and II present in humans. The GnRH-I gene, located on chromosome 8, encodes a pre-hormone essentially involved in reproductive processes, while GnRH-II, found on chromosome 20, exhibits broader tissue expression beyond the brain. GnRH secretion follows a pulsatile pattern, important for sustained gonadotropin release, with the frequency variations occurring in males and females throughout their reproductive cycle.^[7–11]

REGULATION OF GnRH

Norepinephrine, neuropeptide Y (NPY), kisspeptin, and opioid peptides are the key neurotransmitters and neuropeptides that regulate GnRH. Evidence highlights that norepinephrine facilitates GnRH release in female rabbits and is crucial for ovulation. The release of NPY from the hypothalamus in a pulsatile manner further stimulates GnRH release via the α-adrenergic pathway.^[12–14]

A peptide from the KISS-1 gene called kisspeptin is essential for stimulating the GnRH secretion. It binds to the G-protein-coupled receptor 54, is produced in the hypothalamus, and acts on GnRH neurones. Kisspeptin can trigger the production of Luteinizing hormone (LH) from pituitary gonadotrope cells, and studies show that 50%–75% of GnRH neurones have kisspeptin receptors. Mutations in the kisspeptin receptor are mainly linked to idiopathic hypogonadotropic hypogonadism and polycystic ovary syndrome (PCOS).^[15,16] GnRH is also regulated by endothelin-1^[17] and activin A.^[18]

The search for inhibitors of GnRH is extensively studied. Some of which are endogenous opioid peptides that inhibit Preoptic area (POA) GnRH neurones in rats. Evidence proves that chronic treatment with morphine blocked the secretion of GnRH, and also the mRNA levels of POA GnRH were decreased.^[19] It has also been demonstrated that corticotropin-releasing hormone inhibits hypothalamic GnRH secretion. Studies have shown that women with amenorrhoea and high levels of stress experience hypercortisolism, thereby disrupting the reproductive function.^[20,21]

In the quail pituitary, Tsutsui and colleagues’ discovery of a novel RFamide peptide, Ser-Ile-Lys-Pro-Ser-Ala-Tyr-Leu-Pro-Leu-Arg-Phe-NH₂ (SIKPSAYLPLRFamide), inhibited gonadotropin release and hence was named gonadotropin‐inhibitory hormone (GnIH).^[22] Identification of mammalian and primate GnIH peptides that possessed a common C-terminal LPXRFamide (X = L or Q) motif, similar to that of avian GnIH and GnIH-RPs. Mammalian as well as primate GnIH peptides are often referred to as RFamide-related peptides 1 and 3, which inhibit GnRH-elicited gonadotropin secretion in mammals.^[23,24]

POLYAMINES

Polyamines, including putrescine, spermidine, and spermine are small organic cations that contribute to cell growth, differentiation, and gene regulation.^[25–27] In mammals, polyamines are synthesised de novo from amino acids such as arginine, methionine and proline through L-ornithine ^[28–30] which, after absorption by the intestines, is released into the blood circulation.^[31] The biosynthetic pathway of polyamines utilises L-ornithine and S-adenosyl methionine (AdoMet) as substrates to produce the well-known polyamines such as putrescine, spermidine and spermine.^[25–27,32] Microorganisms and plants synthesise cadaverine, agmatine and thermospermine. Low amounts of agmatine and cadaverine are present in certain mammalian tissues.^[33,34] Minority polyamines (thermine, caldine, thermospermine, etc.) have been observed in extreme microorganisms.^[35] The proposed regulatory mechanism of polyamines on gonadotropin-releasing hormone expression is illustrated in Figure 1.

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Figure 1:

Biosynthesis of polyamine.

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Polyamines have been widely reported to control processes of reproduction, including embryo development and testicular function.^[36] Studies have shown elevated ovarian ornithine decarboxylase 1 (ODC1) activity during the prepubertal period in mice and rabbits.^[37] In both immature rats^[38,39] and adult rats^[40] induction of ODC1 was observed by LH and human chorionic gonadotropin, and during the Oestrous cycle, a maximal response of ODC1 is observed during the late proestrus phase.^[41] A study showed induction of ODC mediated by luteinising hormone is necessary for luteinisation and folliculogenesis, and also might regulate steroidogenesis in the ovary.^[37] Polyamines have been shown to participate in the regulation of GnRH and gonadotropin secretion, particularly during the neonatal and infantile periods and have been measured in relevant neuroendocrine areas, i.e., the hypothalamus and pituitary, of the developing rat.^[42] However, their role in regulating GnRH expression/release in adult rodents, and thereby simultaneously altering the gonadotropin production, is not very well understood. Recent studies highlight that the polyamines influence GnRH synthesis, secretion, and receptor activity, thereby playing an integral role in neuroendocrine reproductive control. This emerging perspective offers new insights into the molecular intricacies governing GnRH regulation while offering potential implications for reproductive health and associated disorders.

POLYAMINES AND GnRH

A study suggested that polyamine treatment of the hypothalamus and anterior pituitary obtained from 6- and 15-day-old rats in vitro was able to regulate GnRH, Follicle-stimulating hormone (FSH), and LH secretion.^[42] The same group had demonstrated that Difluoromethylornithine (DFMO) treatment induced delayed puberty of female rats with high FSH levels during the infantile period.^[42]

Our group for the first time showed that polyamine and polyamine-related factors, ODC1, Spermidine (SPD), Spermine (SPM) and Antizyme inhibitor 1 (AZIN1), exhibited a similar pattern of expression to that of GnRH-I in the hypothalamus, ovary and uterus during the oestrous cycle. In the ovary, polyamines and their associated factors, such as ODC1, SPM, SPD and AZIN1. There existed a similarity in the pattern of expression of these with that of steroidogenic factor, StAR, during the oestrous cycle. Thus, suggesting a probable interaction between polyamines and GnRH-I in the hypothalamus and the ovary, thereby regulating ovarian steroidogenesis, folliculogenesis and luteinisation, as well as in the maintenance of oocyte physiology. Similarly, in the uterus, polyamines and their associated expression in the uterine luminal epithelium and glandular epithelium throughout the oestrous cycle suggest that polyamines are vital in regulating and maintaining the uterine physiology.^[1] A study using Indian major carp, Labeo rohita, showed the localisation of GnIH and GnRH in the pituitary during development and suggested their involvement in the regulation of pituitary hormones and reproductive physiological functions.^[43]

Further, endogenous putrescine supplementation to adult female rats revealed an elevation in both GnRH-I expression of the hypothalamus and the levels of GnRH-I in circulation. Supplementation of putrescine resulted in a decline in the expression and release of GnIH. These effects of up-regulating the expression of GnRH-I and down-regulation of the GnIH expression were confirmed by in vitro experiments using GT1-7 hypothalamic cells. The serum levels of gonadotropins, LH and FSH, increased after putrescine supplementation. Putrescine treatment also increased the number of corpus lutea in the ovary and increased the number of uterine glands. Collectively, these rodent studies demonstrated that the hypothalamic-pituitary-gonadal (HPG) axis could be regulated by putrescine through an increase in GnRH-I, LH and FSH levels while suppressing GnIH. Simultaneous effects of putrescine on the regulation of both GnRH-I and GnIH in the hypothalamus have been reported for rodents. However, whether these findings directly translate to humans remains to be established, and further research is required before clinical relevance can be inferred.^[2]

Previous studies described a decrease in the level of polyamine and GnRH-I with age in rodents, with tumour necrosis factor-α (TNF-α) and nuclear factor kappa B (NF-κB) inhibiting GnRH-I during ageing.^[44,45] Studies also showed that polyamines reduce inflammation and apoptosis in the brain, thereby slowing down the ageing process.^[46] Another study reported that dietary spermine supplementation correlated with better cognitive performance in humans.^[47] During ageing and in metabolic disorders, ‘hypothalamic microinflammation’ is observed with increased TNF-α and lactate.^[48,49] Overproduction of TNF-α was reported to inhibit hypothalamic GnRH-I release, causing an age-related decline in GnRH-I levels.^[50] However, the polyamine regulation of GnRH during hypothalamic inflammation in ageing remains elusive, particularly in humans. Recently, our study showed a significant positive correlation between ODC1, SPM, SPD and GnRH-I; SPD, SPM and ODC1 had a significant negative correlation with GnIH protein expression in the hypothalamus. These findings suggest that polyamines might regulate GnRH-I in the hypothalamus during ageing, thus influencing reproductive ageing in rodents. The study further showed treatment with putrescine and agmatine in the presence of high TNF-α and lactate significantly increased GnRH-I both at the peptide as well as mRNA level. Thus, corroborating that polyamines can have neuroendocrine protective roles by rescuing inhibitory effects on hypothalamic GnRH-I by TNFα and lactate, as well as providing homeostasis to GnRH-I synthesis and release in the hypothalamus. This study also showed that treatment of putrescine enhances GnRH-I expression by modulating pathways related to calcium ion signalling, oestrogen signalling, circadian rhythm genes, neuronal differentiation, and neurulation. Recently, we also showed that when young mice when administered with putrescine led to increased ovarian size and a higher number of follicles. Additionally, it exhibited elevated serum GnRH levels and progesterone, along with increased GnRH mRNA expression in rodents. An upregulation of ovarian genes associated with folliculogenesis—such as Fshr, Bmp15, Gdf9, Amh, Star, Hsdb3, and Plaur—and hypothalamic genes linked to puberty onset, Tac2 and Kiss1, after putrescine treatment.^[3,4]

High brain lactate and TNF-α are well-established hallmarks of ageing. In one of our studies, we demonstrated that exposing young mouse brains to supra-physiological lactate levels mimicked age-related metabolic disturbances, leading to the transcriptional repression of GnRH-I. Additionally, a transcriptomic analysis of aged astrocytes revealed that cellular respiration pathways were suppressed, likely contributing to increased lactate production via glycolysis. This lactate is transported into GnRH-I-producing neurones, activates NF-κB signalling and upregulates transcriptional repressors of GnRH-I, ultimately leading to its repression.^[51]

These recent findings from our group provide novel insights into the regulatory role of polyamines in hypothalamic function during ageing, particularly concerning reproductive hormone GnRH expression. These findings suggest that polyamine supplementation can enhance GnRH expression, promote folliculogenesis, and potentially advance puberty onset in young female mice, highlighting the significant role of polyamines in GnRH regulation and reproductive development during ageing.

POLYAMINES IN EARLY DEVELOPMENT AND GAMETOGENESIS

Numerous studies exist related to the effect of polyamines on embryogenesis and implantation. Studies showed ODC1 activity in the embryo likewise increases between the two-cell and early blastocyst stages in the mouse. A similar increase has also been recorded in pigs^[52] and Xenopus. Polyamine-related genes, such as ODC1, Spermidine/Spermine N1-acetyltransferase 1 (SAT1), and AZI, and uterine polyamine contents are significantly up-regulated in the uterus during the early stages of embryo reactivation.^[36,53] A study also showed that polyamines are necessary for placental formation.^[54] ODC1 activity was lower in foetal tissues compared with the placenta, but polyamine content was higher in the foetus and yolk sac relative to the placental compartment.^[55] Reports exist that polyamines have a role in regulating the GnRH secretion from the hypothalamus in the neonatal and infantile periods ^[42] and polyamines also regulate ovarian functions too. Based on the findings described earlier, it is possible to improve PCOS-like conditions in the rat model by treating it with putrescine, a polyamine. It is possible that treatment with putrescine can alter the GnRH expression/secretion pattern from the hypothalamus and thereby can alter the LH/FSH ratio in the cystic females and thus can improve the state of cystic ovaries. Comparative expression patterns observed under different experimental conditions are summarised in Figure 2.

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Figure 2:

Functions of polyamines (putrescine, agmatine, spermidine, spermine, ODC1).

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ALTERNATIVE SPLICING (AS) OF THE GnRH GENE BY POLYAMINES

AS is one of the important mechanisms for increasing the variety of the transcriptome and proteome, especially in mammals.^[56] Several studies show that hypothalamic genes are post-transcriptionally regulated. In a recent study, transcriptome analysis was done to compare the hypothalamic AS events in the differentially expressed genes (DEGs) in the small tail Han sheep, showing approximately 40 AS DEGs in polytocous sheep vs. monotocous sheep in the luteal phase and nearly 39 DEGs with AS events (AS DEGs) in polytocous sheep vs. monotocous sheep in the follicular phase.^[57]

Another study used gilts as a research model to investigate the AS landscape across the hypothalamus-pituitary-ovary axis during distinct pubertal stages and reported approximately 3,000, 6,000 and 9,000 Dehydroepiandrosterone sulfate (DEAS) events in the hypothalamus, pituitary and ovary, respectively, when comparing pre-, in- and post-pubertal phases.^[58] The hypothalamic GnRH gene also undergoes AS, which was first observed in two GnRH-expressing neuronal cell lines, Gn11 and Nucleus lateralis tuberalis (NLT). Reverse transcription polymerase chain reaction (RT-PCR) and RNase protection assays revealed that while NLT cells predominantly express the mature pro-GnRH transcript, GnRH cells mainly produce a truncated splice variant lacking exon 2, which encodes the GnRH decapeptide. This differential splicing, although it requires further validation, likely contributes to the reduced GnRH secretion observed in Gn11 cells.^[59]

The role of polyamine in AS is well established in one finding, where it promotes cyto-protection in normal human CD4⁺ T lymphocytes but not in cancer cells through modulation of RAD51 recombinase A (RAD51A) AS. Specifically, putrescine treatment shifts RAD51A expression from the splice variant lacking exon 4 to the full-length isoform in response to DNA damage. This switch correlated with reduced DNA damage and enhanced resistance to cisplatin-induced apoptosis.^[60]

The author’s group has recently shown that putrescine, a well-studied polyamine, has an impact on the GnRH splicing and the expression of splicing regulatory proteins. The recent study analysed the transcriptome of polyamine-treated vs control, untreated GT1-7 hypothalamic neuronal cells. It was shown that putrescine increased the expression of both the exon-skipped version (GnRH V2) and the full-length GnRH isoform (GnRH V1), with the later showing the higher expression of important AS regulators, such as RNA helicase (DDX26B, DDX6, DDX25), SR protein (SRSF11, SRSF12), pre-mRNA processing factors (PRPF8), and splicing factors 3b (SF3B2). On the other hand, SF3B1 and HnRNPA3 were downregulated. These results imply that putrescine affects the production of GnRH isoforms via two different mechanisms: attenuating elements of the canonical splicing machinery and boosting factors that encourage AS.^[61–65]

CONCLUSION

Hence, the present review highlights the fact that polyamines, particularly putrescine, have evolved to be one of the potent regulators of hypothalamic GnRH expression and release. It was shown that putrescine not only increases hypothalamic GnRH expression in adult cyclic mice but also regulates GnRH expression in aged neurones as well as young mice.

GnRH being an alternatively spliced gene, putrescine has also been shown to regulate the scenario of AS of the GnRH of the hypothalamus. However, further mechanistic studies are required to uncover the mechanism by which putrescine regulates GnRH expression. It is not shown whether other polyamines, such as spermidine and spermine, could be as effective as putrescine in regulating hypothalamic GnRH.

A study showed crosstalk among signalling systems such as adenylate cyclase-, calcium-, and MAP kinase-dependent pathways in the regulation of gonadotropin-induced steroid production in fish ovarian follicles. A study showed exogenous growth hormone-releasing factor induced growth hormone, which further influences the gonadal axis, bringing about early puberty onset in buffalo heifers. The same authors further showed significant correlation between plasma Zn, Fe and FSH concentrations over time and suggested plasma Zn, Fe, Cu and Mn play a vital role in the initiation of puberty in prolific Black Bengal goats. A recent study suggested control of the reproductive cycle in tree sparrows involves the actions of Tsh-β and Dio2/Dio3 regulating seasonal reproduction by modulating GnRH-I via the HPG axis.