A Review on the Role of TRP Channels and Their Potential as Drug Targets_An Insight Into the TRP Channel Drug Discovery Methodologies.

Transient receptor potential (TRP) proteins are a large group of ion channels that control many physiological functions in our body. These channels are considered potential therapeutic drug targets for various diseases such as neurological disorders, cancers, cardiovascular disease, and many more. The Nobel Prize in Physiology/Medicine in the year 2021 was awarded to two scientists for the discovery of TRP and PIEZO ion channels. Improving our knowledge of technologies for their study is essential. In the present study, we reviewed the role of TRP channel types in the control of normal physiological functions as well as disease conditions. Also, we discussed the current and novel technologies that can be used to study these channels successfully. As such, Flux assays for detecting ionic flux through ion channels are among the core and widely used tools for screening drug compounds. Technologies based on these assays are available in fully automated high throughput set-ups and help detect changes in radiolabeled or non-radiolabeled ionic flux. Aurora's Ion Channel Reader (ICR), which works based on label-free technology of flux assay, offers sensitive, accurate, and reproducible measurements to perform drug ranking matching with patch-clamp (gold standard) data. The non-radiolabeled trace-based flux assay coupled with the ICR detects changes in various ion types, including potassium, calcium, sodium, and chloride channels, by using appropriate tracer ions. This technology is now considered one of the very successful approaches for analyzing ion channel activity in modern drug discovery. It could be a successful approach for studying various ion channels and transporters, including the different members of the TRP family of ion channels.

Thyroid Hormones Deficiency Impairs Male Germ Cell Development: A Cross Talk Between Hypothalamic-Pituitary-Thyroid, and-Gonadal Axes in Zebrafish.

In vertebrates, thyroid hormones are critical players in controlling different physiological processes such as development, growth, metabolism among others. There is evidence in mammals that thyroid hormones are also an important component of the hormonal system that controls reproduction, although studies in fish remain poorly investigated. Here, we tested this hypothesis by investigating the effects of methimazole-induced hypothyroidism on the testicular function in adult zebrafish. Treatment of fish with methimazole, in vivo, significantly altered zebrafish spermatogenesis by inhibiting cell differentiation and meiosis, as well as decreasing the relative number of spermatozoa. The observed impairment of spermatogenesis by methimazole was correlated with significant changes in transcript levels for several genes implicated in the control of reproduction. Using an in vitro approach, we also demonstrated that in addition to affecting the components of the brain-pituitary-peripheral axis, T3 (triiodothyronine) also exerts direct action on the testis. These results reinforce the hypothesis that thyroid hormones are an essential element of multifactorial control of reproduction and testicular function in zebrafish and possibly other vertebrate species.

Interaction between thyroid hormones and gonadotropin inhibitory hormone in ex vivo culture of zebrafish testis: An approach to study multifactorial control of spermatogenesis.

Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as of local gonadal signaling systems including sex steroids, growth factors and non-coding RNAs. Among the factors, gonadotropin-inhibitory hormone (Gnih) is a novel RFamide neuropeptide which directly modulates gonadotropin synthesis and release from pituitary, and in the gonads, Gnih mediated inhibitory actions on gonadotropin response of zebrafish spermatogenesis. Thyroid hormones are peripheral hormones which are also known to interact with reproductive axis, in particular, regulating testicular development and function. This study investigated the interaction between Gnih and thyroid hormones in zebrafish spermatogenesis using in vivo and ex vivo approaches. Three experimental groups were established: "control" (non-treated fish), "methimazole" and "methimazole + T4". Fish were exposed to goitrogen methimazole for 3 weeks; T4 (100 µg/L) was added in the water from the second week only in the "reversal treatment" group. After exposure, testes were dissected out and immediately incubated in Leibovitz's L-15 culture medium containing hCG, Gnih or hCG + Gnih for 7 days. Germ cell cysts and haploid cell population were evaluated by histomorphometry and flow cytometry, respectively. Our results showed that hypothyroidism affected germ cell development in basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Hypothyroid testes showed lower amount of spermatozoa, and decreased potency of hCG. We also showed that goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis. This study provided evidences that thyroid hormones are important regulatory factors for hCG- and Gnih-mediated functions in zebrafish spermatogenesis.

Effects of gonadotropin-inhibitory hormone on early and late stages of spermatogenesis in ex-vivo culture of zebrafish testis.

Gonadotropin-inhibitory hormone (Gnih) is known to play a role in the regulation of reproduction in vertebrates by influencing gonadotropin release and synthesis. While the endocrine actions of Gnih have been identified in several species, its paracrine/autocrine effects in the control of spermatogenesis are less defined. We have used ex vivo culture of zebrafish testis to investigate the role of gonadal zebrafish Gnih (zGnih) in the regulation of the spermatogenic process. We used FACScan cell cycle analysis, morphometric quantifications, BrdU incorporation and caspase-3 activity assays as well as measuring 11-Ketotestosterone (11-KT) level in the culture media. FACScan analysis and morphometric quantification results demonstrated direct action of zGnih on basal and gonadotropin (Lh and Fsh)-induced spermatogenesis. Treatment with zGnih (10 nM) significantly decreased the number of G0/G1 cells after 7-days of culture while no significant changes were found in the proportion area of spermatogonia cell types. Investigation of DNA synthesis using BrdU (5-Bromo-2'-Deoxyuridine) labeling showed that treatment with zGnih (10 nM) significantly decreased proliferative activity of type A spermatogonia, while increased the mitotic activity of type B spermatogonia. We also showed that treatment with zGnih (100 nM) completely eliminated 11-KT release induced by 100 ng/ml Fsh. Treatment with zGnih (10 and 100 nM) also inhibited both hCG and Fsh-induced spermatogenesis. These results, plus our previous findings, demonstrate that zGnih produced locally in the testis is a component of a complex multifactorial system that regulates testicular function in zebrafish.

Role of GnRH and GnIH in paracrine/autocrine control of final oocyte maturation.

The control of oocyte growth and its final maturation is multifactorial and involves a number of hypothalamic, hypophyseal, and peripheral hormones. In this study, we investigated the direct actions of the gonadotropin-releasing hormone (GnRH) and the gonadotropin-inhibitory hormone (GnIH), which are expressed in the ovarian follicles, on final oocyte maturation in zebrafish, in vitro. Our study demonstrates the expression of GnRH and GnIH in the ovarian follicles of zebrafish (Danio rerio) at different stages of development and provides information on the direct action of these hormones on final oocyte maturation. Treatment with both GnRH and GnIH peptides stimulated the germinal vesicle breakdown (GVBD) of the late-vitellogenic oocyte. Both the GnRH and GnIH treatments showed no significant change in the caspase-3 activity of pre-vitellogenic and mid-vitellogenic oocytes, while they displayed different responses in the late-vitellogenic follicles. The GnRH treatment increased caspase-3 activity, whereas the GnIH reduced caspase-3 activity in the late-vitellogenic follicles. We also investigated the effects of GnRH and GnIH on the hCG-induced resumption of meiosis and caspase activity in vitro. GnRH and GnIH were found to have a similar effect on the hCG-induced resumption of meiosis, while they showed the opposite effect on caspase-3 activity. Furthermore, we investigated the effects of concomitant treatment of GnRH and GnIH peptides with hCG. The results demonstrated that the presence of both GnRH3 and GnIH are necessary for the normal induction of final oocyte maturation by gonadotropins. The findings support the hypothesis that GnIH and GnRH peptides produced in the ovary are part of a complex multifactorial regulatory system that controls zebrafish final oocyte maturation in paracrine/autocrine manner working in concert with gonadotropin hormones.

Paracrine/autocrine control of spermatogenesis by gonadotropin-inhibitory hormone.

Control of testicular development is multifactorial and involves a number of hypothalamic, hypophyseal and peripheral hormones. Here, we investigated direct action of zebrafish gonadotropin-inhibitory hormone (zGnih) which is expressed in the testis, on spermatogenesis in zebrafish, in vitro. Treatment with zGnih at the lower doses (10 and 100 nM) inhibited gonadotropin-induced spermatids/spermatozoa (SPD/SPZ) production. However, at the highest dose (1000 nM), zGnih increased basal number of SPD/SPZ and showed paradoxical effect. The effects of zGnih on testosterone and SPD/SPZ production was blocked in the presence of androgen receptor antagonist, flutamide (FLU). A number of transcripts were also measured to better understand zGnih mechanisms of action on zebrafish spermatogenesis. Our results provide strong support for the hypothesis that locally produced zGnih is a component of the complex multifactorial system that regulates testicular development and function in adult zebrafish, in part, by changes in testicular steroidogenesis and regulation of gonadotropin-induced response.