Gonadotropins are glycoprotein sex hormones regulating development and reproduction and bind to specific G protein–coupled receptors expressed in the gonads. Their effects on multiple signaling cascades and intracellular events have recently been characterized using novel technological and scientific tools. The impact of allosteric modulators on gonadotropin signaling, the role of sugars linked to the hormone backbone, the detection of endosomal compartments supporting signaling modules, and the dissection of different effects mediated by these molecules are areas that have advanced significantly in the last decade. The classic view providing the exclusive activation of the cAMP/protein kinase A (PKA) and the steroidogenic pathway by these hormones has been expanded with the addition of novel signaling cascades as determined by high-resolution imaging techniques. These new findings provided new potential therapeutic applications. Despite these improvements, unanswered issues of gonadotropin physiology, such as the intrinsic pro-apoptotic potential to these hormones, the existence of receptors assembled as heteromers, and their expression in extragonadal tissues, remain to be studied. Elucidating these issues is a challenge for future research. Receptor specificity is due to unique beta subunits encoded by follicle-stimulating hormone beta-subunit gene (FSHB) and luteinizing hormone beta-subunit gene (LHB), expressed in the pituitary. Choriogonadotropin beta-subunit genes (CGBs) are expressed in trophoblast cells and placenta3.Gonadotropins are glycoprotein hormones regulating development and reproduction via targeting gonadal cells expressing specific G protein–coupled receptors. Gonadotropins are dimers having structural similarities and an identical common alpha subunit, and the beta subunit conserves about 70 to 80% of amino acid sequence identity in humans4. In the female, FSHR expression in granulosa cells is a requisite for sustaining the monthly ovarian follicle recruitment and subsequent FSH-mediated events, such as oocyte maturation, selection, and estrogen synthesis5. LH modulates proliferative and anti-apoptotic signals in granulosa cells, accompanied by androgen production in theca cells, and culminates in the induction of the dominant follicle ovulation and progesterone production by luteal cells6. During the first trimester of pregnancy, human chorionic gonadotropin (hCG) replaces the luteotrophic role of LH in sustaining the demand for increasing levels of progesterone. Chorionic gonadotropin is encoded by a cluster of six genes, embedding two pseudogenes7, resulting in several slightly different isoforms and glycosylation variants acting through the LHCGR8. Although hCG glycoforms have different potencies in activating LHCGR-mediated signaling pathways9, the specific physiological functions of these variants, if any, are uncertain. It was hypothesized that they evolved in humans to optimize fetal brain development10.During the fertile age, the action of gonadotropins is sex-specific and its secretion is regulated by a feedback mechanism depending on levels of sex steroid hormone. In the male, FSH binds its receptor (FSHR) expressed in Sertoli cells, where the hormone exerts trophic functions fundamental for sustaining gamete production and maturation. In contrast, LH binding to its receptor (LHCGR) stimulates the synthesis of testosterone by Leydig cellsThe physiological functions of gonadotropins are exerted via multiple signaling cascades simultaneously activated in the target cells. A limited number of these pathways were known for decades, but an increasing body of knowledge emerging from recent literature is uncovering a more complex and complete view of the action of these hormones. Therefore, we provide an update on the classic view of gonadotropin physiology, reviewing recent advancements as well as unanswered questions.αs protein/adenylyl cyclase-dependent cAMP/protein kinase A (PKA) pathway. Historically, this pathway was studied using immunoassays to quantify cAMP accumulation in target cells thanks to the use of specific inhibitors and activators11. It was discovered that this pathway is fundamental for regulating steroidogenic events in gonadal cells12, as already known for the functionally similar steroidogenic cells of the adrenal gland13. An exception is provided by adult Sertoli cells, which cannot synthesize sex steroid hormones, and the cAMP/PKA pathway activated therein results in FSH-dependent trophic signals, sustaining cell metabolism and viability14. However, the activation of this intracellular signaling pathway may be triggered by all gonadotropins in target cells, where the intracellular cAMP increase is linked to other events such as trophic effects, mitotic functions, and sometimes apoptosis14. Downstream PKA, the cascade of kinase activations results in the extracellular-regulated kinase 1 and 2 (ERK1/2). The cAMP-responsive element-binding protein (CREB) phosphorylation is activated as well, inducing transcription of target genes, such as the steroidogenic acute regulatory protein StAR15. These common pathways modulating the steroidogenic signals are regulated in a cell- and developmental stage-dependent manner, and many newly discovered molecules and pathways were found to support steroid synthesis. For instance, protein kinase C (PKC) may amplify the cAMP-dependent steroidogenesis in both granulosa and Leydig tumor cells16, but another research documented the opposite effects of PKA and PKC on progesterone synthesis in ovarian steroidogenic cells17. Interestingly, a recent study found that, in granulosa cells, lisosphingolipids may induce cAMP-independent CREB phosphorylation, which, however, is not linked to the activation of steroid synthesis18. These data suggest that steroidogenesis is strictly under the control of gonadotropins and that the phosphorylation of a transcription factor per se is not enough to trigger the transcription of key enzyme-encoding genes. Moreover, it was recently demonstrated that the FSH-dependent activation of steroidogenic signals and folliculogenesis might be impinged by salt-induced kinases, indicating the relevance of these enzymes in regulating ovarian functions19. On the other hand, in luteal cells, progestational signals specifically regulated by LH via activation of the cAMP/PKA pathway involve the Wingless and Int-1 (Wnt) pathway components glycogen synthase kinase-3β and β-catenin and increase the acute progesterone synthesis in response to gonadotropin stimulation20. These data are representative of cell-dependent, complex signaling networks interacting with the classic cAMP/PKA pathway for supporting the synthesis of steroid hormones. New insights into the modulation of the steroidogenic pathways were provided by experiments performed using Leydig cell lines, treated with recently developed allosteric modulators of gonadotropin receptors. This experimental setting revealed that β-arrestin 2, known to be involved in G protein–coupled receptor (GPCR) internalization and sustained signal transduction21, may act as a negative modulator of testosterone synthesis22. Interestingly, steroidogenic signals can be induced by cAMP-independent mechanisms, at least in Leydig cells, where the LH-dependent phosphorylation of ERK1/2 and CREB may occur via activation of the Rous sarcoma oncogene-encoded protein (SRC)23 and epidermal growth factor receptor24.One of the best-known intracellular signaling cascades activated by gonadotropins is the G2+) increase, and the recruitment of the adaptor protein “APPL1”31. The overlapping of FSHR- and LHCGR-dependent intracellular signaling pathways would be due to structural similarities between the two receptors as an inheritance persisting during the evolution of an encoding gene from an ancient common ancestor sequence32. Interestingly, mutations conferring constitutive activity to the FSHR may even support sex steroid–independent spermatogenesis34, suggesting that certain gonadal functions may be supported by the activation of overlapping pathways.The two gonadotropin receptors have common signaling pathways. Besides triggering the aforementioned cAMP/PKA pathway, both membrane receptors are known to trigger the activation of p38 mitogen-activated protein kinase (MAPK) pathway, an intracellular calcium ion  pathway51. Gonadotropins can be naturally glycosylated to produce variants with physiological relevance as well as artificial compounds, synthesized for clinical purposes, with different glycosylations and pharmacological impact. The alpha subunit carries two carbohydrate chains joined to asparagine residues (N-linked glycosylation), and one and two are linked to the LHβ and FSHβ, respectively. Two N-linked glycosylation sites are also present in the hCGβ, which, in addition, has four glycosylated serine/threonine residues (O-glycosylation)52. This classic view has been revisited, especially in the last decade, thanks to studies deepening our understanding of the glycosylation on the function of FSH53, LH54, hCG55, and even of the related glycoprotein thyroid-stimulating hormone (TSH)56. For instance, the existence of several hCG glycoforms is well known, was reviewed in 2016, and was considered to have a pathophysiological sense57. It was proposed that carbohydrate chains on hCG may be used to predict pregnancy outcomes as a marker of pathologies and infertility treatment62. During the first trimester of pregnancy, the quantitative and qualitative production of hCG glycoforms is exceptionally variable, starting with hyperglycosylated forms (H-hCG) of trophoblastic origin, followed by less glycosylated isoforms63. Moreover, H-hCGs were found in the serum of patients affected by certain tumors64. Given these observations, it was supposed that H-hCG, rather than being an activator of progesterone synthesis65, would play a key role in inducing proliferative signals regulating trophoblast invasion and angiogenesis during the first two weeks of pregnancy as well as tumor growth66. This action would be exerted via cross-interaction between the hormone and the tumor growth factor beta (TGF-β) receptor67. However, this issue is controversial since another study found no TGF-β receptor activation by hCG and, instead, suggested the presence of growth factor contamination in the gonadotropin preparations68. Less glycosylated, “classic” hCG molecules have a shorter half-life than H-hCG8 and higher potency for LHCGR activation9. These features characterize hCG as a hormone optimized for inducing maternal and fetal steroidogenesis, which is fundamental for supporting massive progesterone production by the corpus luteum and the placenta, for estrogen production, and for triggering the masculinization of the male fetus69. hCG does also exert a TSH-like action, inducing the production of thyroid hormones acting on the TSH receptor (TSHR)70. Although the TSH-like actions of hCG require further research, a 2009 study suggested a link between thyroid autoimmunity and miscarriage71. However, conflicting data raise concerns about this hypothesis72. Finally, gestational hyperthyroidism has been linked to the expression of TSHR gene variants with hCG hypersensitivity73.It was previously suggested that the incorporation of oligosaccharides in the structure of gonadotropins impacts intracellular signaling cascades activated by the hormonesin vitro demonstrated that the glycosylation pattern of these molecules might modulate the intracellular signaling and gene expression9. Similarly, changes in the glycosylation and composition of glycans attached to FSH and LH, related to the stage of the menstrual cycle, were found in the serum of women of fertile age75. Thus, it cannot be excluded that glycoform-specific intracellular signals can be activated76. For instance, poorly glycosylated FSH molecules are more active in vitro than those fully glycosylated77 and this observation was repeatedly confirmed both in vitro79 and in mouse models82. Together with FSH glycoforms, di- and tri-glycosylated LH molecules were supposed to contribute differently to the regulation of natural ovarian cycles54 but these data need to be confirmed. To conclude, steps forward have been taken to elucidate molecular aspects related to gonadotropin glycobiology and the issue nourishes increasing interest of scientists and clinicians using these hormones as drugs for infertility treatment83. However, a clear-cut demonstration of its real impact on human physiology remains to be determined. Actually, the data available indicate that these glycoforms modulate short-term intracellular effects in vitro86, which are flattened in the long term86 and are extremely difficult to evaluate in humans without performing clinical trials with very large sample sizes87.Although the debate over the pathophysiological impact of hCG glycoforms is far from being fully clarified, findings 88. The binding of the ligand triggers a two-step activation mechanism, including the reshaping of the hormone conformation after contacting the LRR domain89. These events are followed by the interaction of the alpha and beta subunits with a sulfated tyrosine residue within the hinge region90. This process leads to the stabilization of ligand–receptor complexes and the inhibition of the agonistic activity of the hinge region50. As described above, these events are needed for triggering the Gαs protein coupling to the gonadotropin receptor and activation of the downstream cAMP/PKA pathway, which are preceded by conformational rearrangements of the transmembrane regions91. However, receptors may exist in multiple active conformations, leading to several signaling cascades simultaneously activated by coupling to other G proteins and intracellular interactors, triggering a network of simultaneous intracellular pathways92. Thus, particular signaling patterns may be induced by ligands displaying agonist, antagonist, or inverse agonist activities (or a combination of these) at the same GPCR, depending on the downstream endpoint evaluated, via induction of specific spatial conformations of the receptor molecule93.All GPCRs are activated upon hormone binding to their orthosteric site, which, in the case of gonadotropin receptors, is located in a wide horseshoe-shaped extracellular domain with leucine-rich repeats (LRRs)FSHR gene (2039A>G), leading to the amino acid change of asparagine to serine at position 680 at the intracellular portion of the protein . This SNP modulates the kinetics of cAMP activation, ERK1/2 and CREB phosphorylation, and synthesis of sex steroids94 and is associated with the gonadal response to FSH in males and females96.Although these features may be due to the aforementioned hormone glycosylation variants, another mechanism that may alter GPCR signaling may be provided by single-nucleotide polymorphisms (SNPs) at the receptor level. This is the case of the common nucleotide change at position 2039 of the 97 and modulate desensitization, internalization, and recycling. They also counteract Gαs protein coupling to the receptor98 and upregulate ERK1/2 and AKT phosphorylation99. Moreover, the action of β-arrestins is susceptible to the phosphorylation pattern of the intracellular carboxyl-terminal end of GPCR, which differentially contributes to the recruitment of the scaffold protein, trafficking, and specific intracellular localization100 of ERK1/2 activation102. These data may explain why overexpression of β-arrestins is linked to attenuation of intracellular cAMP increase103 and may be associated with cell proliferation46. In particular, given the positive impact of β-arrestin functioning on tumorigenesis and cancer cell growth104, they have been suggested as a therapeutic target106 and promoted the development of in vitro systems for studying their functions107. Notably, β-arrestin–induced pERK1/2 activation occurs later but is more sustained than that triggered by the Gαs protein108, revealing that intracellular kinase cascades may be targeted via distinct pathways and kinetics, a molecular mechanism likely modulating specific cell metabolic events. However, β-arrestins and G proteins may also cooperate via the formation of complexes linked to the receptor, leading to temporally sustained cAMP signaling activated from internalized cellular compartments21. These molecular assemblies may also mediate the inhibition of G protein signaling, depending on the spatial conformation of the GPCR–β-arrestin complexes109. Therefore, elucidating the mechanisms biasing gonadotropin receptor signaling is of crucial relevance for developing new therapeutic approaches to certain diseases.Beta-arrestins are scaffold proteins that directly interact with the gonadotropin receptors110. Increasing interest arose around allosteric ligands, which bind the receptor in a site different from the hormone-binding site111. According to the mode of action, these molecules are grouped into classes defined as neutral, negative (NAMs), or positive (PAMs) allosteric modulators. All of these molecules require gonadotropin binding to the receptor for exerting their action111 and modulate receptor-mediated signaling in the presence of the natural ligand. Interestingly, compounds acting via modulation of gonadotropin receptor assembly as homo/heteromers also have been described112. Among allosteric agonists, thiazolidinones are known to bind the FSHR transmembrane domain inducing the activation of Gαs protein-dependent pathways, similarly to FSH. However, compounds with preferential Gαi protein stimulatory activity also have been described114. Benzamides and dihydropyridines are known to have PAM activity at nanomolar concentrations for both the LHCGR and FSHR, activating cAMP in the presence of the bound ligand116. Tetrahydroquinolines belong to the NAM group for both gonadotropin receptors inhibiting cAMP and progesterone, but not estradiol, production and maturation of the ovarian follicle in vivo119. Finally, compounds with antagonistic activity for FSHR were found to block FSH-induced steroid synthesis121, similarly to what was recently found for the LHCGR122. Taken together, novel molecules with agonistic activity and able to differentially activate intracellular signaling are promising tools to be potentially used for optimizing or personalizing treatments of human infertility. Instead, molecules with NAM activity at the gonadotropin receptor, inhibiting the follicle growth without impairing steroid production, could become an option for contraceptive purposes.In the last decade, small-molecule chemical compounds able to bind and modulate FSHR-mediated signaling have been developed91. For instance, antibodies binding the FSHβ subunit were found to prevent the binding to the receptor and have protective effects against bone loss in ovariectomized mice123. Antibodies targeting a placental bovine hCG-like LH (equine chorionic gonadotropin), with both FSH and LH activity in other mammals, would be responsible for forming hormone–antibody complexes with increased β-arrestin–dependent signaling and activity in vivo125. New monoclonal and polyclonal compounds with agonistic activity for gonadotropin receptors and targeting the hinge region are currently under study128 and may represent an alternative approach to allosteric chemicals for modulating gonadal functions. Among these immunoglobulins, nanobodies against the FSHR, with small molecular weight and low predicted immunogenicity in humans, can inhibit cAMP production129. In conclusion, immunoglobulins are promising compounds for future therapeutic purposes, such as infertility131 and cancer132 treatment.New and exciting insights were provided by antibodies modulating selective activation of GPCRs via stabilization of particular hormone or hormone receptor conformations133, and the elucidation of their physiological significance was addressed recently134 but remains mostly unclear. The current knowledge of gonadotropin receptor post-endocytotic processing indicates that these molecules are internalized and either recycled or directed to degradation after ligand binding and G protein activation. The activated receptor then is phosphorylated by GPCR kinases (GRKs) and recruits β-arrestins, leading to G protein uncoupling, desensitization, and internalization via the formation of clathrin-coated pits135. Recent studies have found that β-arrestin–mediated GPCRs internalization may also occur without uncoupling to G proteins, forming a molecular super-complex mediating sustained signaling136. Interestingly, most of these discoveries were provided by functional studies using in vitro transfected models, overexpressing the human FSHR and LHCGR, or rodent receptors, revealing that the internalization of human molecules is slower than that of murine receptors and is due to species-specific amino acids in the intracellular loop of the transmembrane region and the carboxyl-terminal tail137.Mechanistic models explaining GPCR pathways of desensitization and recycling have greatly progressed in recent years. These functions have been known for decades138. Therefore, the endocytotic pathways may sort the fate of receptors impacting their density at the cell surface and the spatial and temporal regulation of intracellular signaling139. One of the most studied pathways involves the formation of early endosomes (EEs). These structures are characterized by the presence of the Ras-related protein 5 Rab-5 (RAB5) and consist of fusion vesicles routing from the plasma membrane and the trans-Golgi network140. The embedding of GPCRs in EEs is the first step of a process directing receptors to two possible fates; the first one is the degradative pathway, which is triggered by the replacement of RAB5 with RAB7, thus trafficking these units to form multi-vesicular bodies preceding protein degradation in the lysosomes. This is the pathway controlling receptor desensitization, which occurs after persistent stimulation of the GPCRs139. Trafficking-defective receptors due to mutations causing protein misfolding are known and may provoke reproductive dysfunctions falling within a wide range of phenotypes138. Interestingly, defects of genes involved precisely in EE functioning were recently associated with dysregulation of GPCR signaling and linked to pathological conditions, such as polycystic ovary syndrome. For instance, perturbation of RAB5- or the clathrin-binding protein (DENND1A)-encoding gene expression in theca cells may result in over-activation of androgen synthesis and subsequent increased susceptibility to the disease141. Another pathway regulating the fate of receptors involves the ubiquitin or RAB4 protein, rerouting EEs to degradation or to recycling endosomes, respectively. The latter route depends mostly on a so-called “PDZ ligand” binding sequence at the carboxyl-terminal tail of LHCGR142, and FSHR recycling may also occur via alternative pathways133. In this case, a crucial role is played by a palmitoylated cysteine at the C-tail, which is required for maintaining proper FSHR recycling, preventing excessive degradation and, likely, impairment of the cell signaling143.GPCR internalization is followed by the activation of a recycling pathway mediated by different endosomes or by the degradation via lysosomesαi-interacting protein C-terminus” (GIPC) with the PDZ protein binding domain100. VEE differs from the EE in that the former lacks RAB5 and is smaller, but in vitro removal of the PDZ domain from LHCGR re-routes the receptor to the EE100, inducing sustained ERK1/2 phosphorylation and revealing that these recycling pathways are interconnected. Interestingly, a similar behavior was observed for FSHR in vitro, using GIPC knockout without the PDZ domain, demonstrating the involvement of APPL1 in the receptor recycling through VEEs145. Indeed, APPL1 may interact with GIPC146 and sustains FSHR-mediated ERK1/2 phosphorylation, as well as AKT activation and recruitment of the 14-3-3τ adapter protein147, through a subpopulation of VEEs positive for APPL1. Interestingly, this molecule is recruited upon cAMP/PKA activation occurring via the FSHR and LHCGR itself148 and depletion of VEE formation results in cAMP inhibition, as demonstrated for other GPCRs149. However, PKA (and VEE) blockade by a specific inhibitor did not evoke similar effects, at least for LHCGR, which can induce sustained cAMP signaling, indicating a dual, receptor-specific effect of APPL125. These data may be relevant for elucidating the whole functions exerted by gonadotropin receptors and their physiological impact. For instance, sustained LHCGR-mediated cAMP signaling would be required to unlock the oocyte from meiotic blockade150, demonstrating its effects on reproduction. In summary, gonadotropin receptor trafficking, being much more than a pathway for recycling these molecules to the cell surface, plays a crucial role in sustaining prolonged intracellular signaling , where LHCGR is routed through the interaction of the so-called “Gignaling .151. Despite this knowledge, the fact that the two gonadotropins act on the same receptor and the difficulties of dissecting these differences in clinical studies152 support the common belief that they are two clinically equivalent hormones. Thus, commercial preparations consisting of FSH, which may be supplemented with LH or hCG or both, were developed153 under the assumption that hCG would provide “LH activity”154 during the follicular phase of the menstrual cycle in inducing oocyte maturation and ovulation153. In the last decade, several studies elucidating specific roles of LH and hCG were published and reviewed157, pinpointing the differences between the two gonadotropins.LH and hCG are distinct molecules that have different functionsCGB genes originated in primates likely by loss of the original stop codon and repeated duplication from an ancestral LHB gene159. More generally, CG molecules exist first in primates160 but are missing in other mammals traditionally used to evaluate the action of hCG, such as mice163. Indeed, historically, LH action was typically studied using hCG in rodents. This is indicative of hCG binding capability to non-primate receptors that are structurally similar to primate LH/CG receptors164. Thus, it appears that in humans the complexity of the LHβ/hCGβ-encoding gene cluster is greater than that of other primates. It was suggested that this complexity might serve for supporting a more complex mechanism of placentation and highly energy-demanding brain development in humans10 but this remains an unproven claim. In any case, this complexity indicates a specialization occurring uniquely in the LHCGR, which, in contrast to FSHR, discriminates between two ligand-specific signals and physiological functions. This exclusive feature would rely mostly on the region encoded by the LHCGR gene exon 10, which may recognize and distinguish the molecular interaction between LH and hCG, modulating different hormone receptor conformations and intracellular signaling166. The role of the exon 10–encoded receptor domain was highlighted by a clinical case describing a boy with impaired development of secondary sexual characteristics due to a naturally occurring deletion of the LHCGR exon 10151. Although the phenotype at birth was clearly male, this patient presented with primary hypogonadism, which could be rescued by treatment with hCG, suggesting a possible action of the maternal choriogonadotropin inducing fetal sex determination but a lack of function of the high LH levels after birth. These data were corroborated by the discovery that the LH receptor sequence of the New World monkey Callithrix jacchus naturally lacks the exon 10–encoded domain. In this primate species, the pituitary produces an hCG-like molecule and not LH, thereby replacing the role of LH168 in other species in which exon 10 is present.LHβ and hCGβ have a common evolutionary origin since in vitro and clinical data indicate that these hormones could be equivalent in inducing testosterone synthesis and spermatogenesis170, reflecting the primary androgenic role of Leydig cells, different results were found in in vitro models of ovarian cells172. The two molecules display different intracellular signaling  cannot discriminate qualitatively between the two human ligands170. This discrepancy between human and mouse receptor functioning should be due to LHCGR key residues involved in hCG/LH discrimination166, which are missing in the Lhr176. In any case, data from human cells support the different physiological roles of the two hormones, which would consist of potent progestational effects mediated by hCG and a fine-tuned regulation of life signals required for ovarian follicle growth and maturation for LH3. Moreover, clinical hormone-specific effects may be evaluated by meta-analyses of studies analyzing large datasets, oppositely to what other studies performed in a clinical177 or in vitro86 context. A proper sample size is required for dissecting the effects of the two hormones in vivo, which indeed turn out to be different. Although the addition of hCG to FSH treatment for assisted reproduction may optimize the number of oocytes retrieved, suggesting an effect positively modulated by steroids, LH could be beneficial for the pregnancy rate as an indirect measure of better oocyte quality152. These data may provide helpful insights for developing personalized protocols for assisted reproduction in specific categories of patients. For instance, poor-responder women178 or those requiring luteal support180 might benefit from the addition of LH and hCG, respectively, during the management of an assisted reproductive cycle179. On the other hand, gonadotropin-releasing hormone (GnRH) agonists are now the alternative option to hCG to prime ovulation and reduce the risk of ovarian hyperstimulation syndrome182, but there are no clinical data comparing the effects of LH and hCG in men.Evolutionary issues suggest that LH and hCG exert different roles, a concept confirmed by the nature of their physiological functions. Although both ignaling , indicat184, polymorphic allelic variants are commonly widespread among the human population, achieving allele frequencies greater than 1%185. To date, these variants may be identified by genetic screening at relatively low costs; in some instances, they are becoming relevant for developing individualized clinical treatments.More than two decades of studies focusing on polymorphisms of gonadotropins and their receptors revealed the existence of allelic variants linked to specific reproductive phenotypes or increased risk of developing related pathologies. In contrast to mutationsFSHB and FSHR genes are promising candidates to be characterized for pharmacogenomic approaches since they impact the ovarian response to hormone stimulation186. Among the roughly 20 SNPs found within the FSHB gene, the only one demonstrated to have physiological significance and clinical impact so far is the guanidine (G) to thymidine (T) variation 211 nucleotides upstream of the start codon 47. In men, the T homozygosity is associated with lower serum FSH levels and testicular volume than other haplotypes187 and decreased sperm quality188. However, similar findings were not always replicated by other studies evaluating spermatogenic potential and Sertoli cell number189. Even the opposite results were found in women190, suggesting possible sex-specific effects of this SNP. In females, the T allele decreases the risk of developing endometriosis, delayed age of menopause, and longer menstrual cycles than the G allele191. At the same time, other studies found an association between the T allele and the FSH and LH levels, idiopathic infertility, and worst clinical assisted reproduction outcome193. These data indicate a lower transcriptional activity of the T allele of the FSHB promoter SNP194, which would modulate the circulating levels of the hormone, thus impacting ovarian response.Genetic variants of FSHR mutations have been described within the receptor regions encoded by exons 7 and 10184. Loss-of-function FSHR mutations may lead to pathological conditions, such as amenorrhea, in women186 and may be linked to infertile or subfertile phenotypes in men196. FSHR mutations overall are rare, and several FSHR SNPs may have been characterized and analyzed for their associations with the gonadal response. Among the best-known common FSHR variants, the rs1394205 (c.-29G>A) in the FSHR promoter, as well as the two exon 10 SNPs rs6165 (c.919G>A) and rs6166 (c.2039G>A) usually in linkage disequilibrium, have been associated with the gonadal response to FSH. In particular, the rs6166 variant is linked to the asparagine-to-serine amino acid change at position 680 of the protein chain (p.N680S) and is located in the C-terminal region of the receptor. This substitution adds a potential phosphorylation site, increases early intracellular signaling, and impacts the kinetics of steroid synthesis94. In practice, homozygous individuals carrying the amino acid serine are less “sensitive” to FSH and show higher serum FSH levels and longer menstrual cycles than pN680S N homozygous women197. A role for FSHR SNP in male fertility has also been proposed198. Cases of idiopathic infertility related to this SNP were described in men199. However, these findings were obtained after analysis of the cumulative effects of the FSHR p.N680S SNP and other SNPs, revealing the overall mild but detectable impact of the polymorphism on male reproduction199. In men, the impact of the SNP rs6166 in modulating the action of FSH was eventually characterized by a pharmacogenetic study where sperm DNA fragmentation of idiopathic infertile patients improved after prolonged treatment with the gonadotropin96. Therefore, the p.N680S phenotype may be a marker of sperm quality.A number of activating and inactivating FSHR exon 10 SNPs should take into account the expression level of the receptor protein given that the polymorphism c.-29G>A modulates the transcriptional activity of the gene200 and is linked to genotype-specific gonadal functions in both males202 and females205. In particular, associations between reproductive parameters and the c.-29G>A SNP were found when analyzed together with the p.N680S FSHR polymorphism201, revealing the cumulative effects of these hot spots. However, the opposite results, which likely indicate a weak clinical impact of the SNP per se, have also been reported208. Further studies using larger datasets are required to highlight the impact of this genetic variant on fertility. Therefore, the combined evaluations of several FSHR and FSHB SNPs are determinant to indicate how the whole haplotype is linked to specific endocrine phenotypes.Studies analyzing the allele frequencies of 199. These analyses should account for limitations intrinsic to each study. Different results due to ethnicity-related209 or sample size210 issues may occur, leading to the requirement of independent confirmation of the findings. Other limitations may be linked to the study design since weak selection criteria and different or unspecified primary endpoints provided non-comparable results199. Extensive interventional studies are needed to more deeply explore the therapeutic benefit of a pharmacogenomic approach to gonadotropin treatment. Nevertheless, after about two decades of small studies on markers of the gonadal response, scientific support for the development of a pharmacogenetic approach to personalized hormonal treatments is now available. Given the dramatic cost reduction for genetic testing in recent years211, conditions are now favorable for SNP analysis before clinical treatments of infertility.Although each FSHR SNP was linked to particular endocrine phenotypes in some studies, the combined analysis of several alleles revealed the real impact of specific haplotypes in increasing the risk of infertility14. Gonadotropins are classically linked to proliferative and survival signals, reflecting their roles in supporting gametogenesis. On the other hand, these molecules upregulate the phosphorylation of mitogenic/anti-apoptotic signals induced via ERK1/2 and AKT and through activation of several different intracellular pathways, including the positive impact on steroid hormone production, supportive of reproduction. In fact, convincing data confirmed that FSH and LH mediate signals required for sperm and somatic cell life in the male gonad212. Interestingly, the textbook knowledge of ovarian physiology is that follicular dominance is a process regulated by pro-apoptotic stimuli occurring after the decrease in serum FSH levels. This event would be responsible for declining proliferative signals occurring in individual follicles, which become atretic. The proliferative effect of gonadotropins is widely described in the scientific literature, where a number of studies describe how FSHR and LHCGR may activate life signaling pathways and, in certain cases, have even a tumorigenic potential5. However, pro-apoptotic stimuli delivered by gonadotropins via cAMP-related pathways were also described215. These effects would be triggered by relatively high intracellular levels of the second messenger, likely resulting in p38 MAPK and p53 activation217 together with steroidogenic signals218 and inducing cell death. From this point of view, the pro-apoptotic potential of gonadotropins would rely on the capacity to increase cAMP, a property previously demonstrated for FSH and hCG in certain experimental conditions in vitro172. In particular, persistently high FSHR expression levels and the lack of steroid hormones could be linked to insufficient inhibition of death signals172, a possible explanation for the lack of consistent human ovarian steroidogenic cell lines expressing this receptor46. This condition is hardly obtainable in vivo in the ovary, where the FSH-induced signals are translated to estrogen production and FSHR is transitorily expressed in the follicles. On the other hand, steroids activate anti-apoptotic signals219, as obligatory regulators of folliculogenesis220, even inducing multi-follicular maturation during assisted reproduction222, where relatively high gonadotropin doses are administered. However, death signals delivered through FSH may play an essential role in the ovary. They could be involved in the selection of the dominant follicle, inducing steroidogenesis and atresia of those follicles expressing relatively high levels of FSHR215. The activation of cAMP is due to the preferential coupling to the Gs protein when FSHR is maximally expressed223 in granulosa cells at the early/mid-antral stage of the menstrual cycle. Interestingly, oocyte maturation might depend on the capability of the membrane G protein–coupled estrogen receptor (GPER) to reprogram cAMP/death signals, linked to FSHR, into proliferative stimuli. The anti-apoptotic role of GPER in the ovary is exerted through the physical interaction with the FSHR, leading to the formation of heteromers activating the AKT pathway upon FSH binding215. These data indicate that the temporary succession of pro- and anti-apoptotic signals is orchestrated, at least in part, by interacting gonadotropin and steroid hormone membrane receptors, playing an essential role in female reproduction. Further studies are required to understand how these molecules act in concert to regulate folliculogenesis. Membrane receptor heteromers might be markers of ovarian cell proliferation215 to be targeted by drugs for infertility and cancer treatment.Although steps forward have been made in the elucidation of gonadotropin physiology over the last three decades, several unknown aspects of their function remain. One of the most underrated issues related to gonadotropin signaling is the activation of pro-apoptotic signals224. It is reasonable that GPCRs interact with molecules having an overall similar structure, as previously demonstrated for receptors of other pituitary225 and non-pituitary226 hormones. In particular, FSHR–LHCGR heteromers were demonstrated in vitro228, where they are classically overexpressed in transfected cell lines and analyzed using imaging methods230. Recently, the GPER has been indicated as a novel interacting partner of both FSHR and LHCGR in ovarian cells215. The physiological implications of these heteromers are not fully understood. It was proposed that they mediate GPCR functional diversity, highly relevant for human physiology231. In particular, they should be involved in the development of competent oocytes during the normal menstrual cycle, reprogramming gonadotropin receptor–mediated death signals in proliferative stimuli232. Impairment of hormone-dependent signaling due to FSHR–LHCGR heteromer formation was reported in the transiently transfected human embryonic kidney (HEK293) cell line233, further providing the rationale for the assembly of heteromers. However, the existence of naturally formed gonadotropin receptor heteromers, as a general feature of GPCRs, is not widely accepted226. In fact, this concept may be weakened by the difficulties in demonstrating heteromers in primary cell cultures, unspecific signals, or elevated background noise due to biosensor-tagged receptor overexpression obtained in an artificial system in vitro as well as the doubt that agonist-induced signals can be generated by heteromers or conformational changes within preassembled groups of receptors234. Therefore, the fact that gonadotropin receptor heteromers may occur in vivo is under debate. A step forward was taken by obtaining data in genetically modified mice coexpressing binding-deficient and signaling-deficient LH receptors235. These experiments were based on a concept previously developed and demonstrated in vitro; binding-deficient receptors triggered cAMP activation through the interaction with a receptor, defective in signal generation, bound to hCG236. Even in mice, LH was able to trigger the activation of the cAMP/PKA intracellular signaling cascade via intermolecular cooperation between the two receptors235. This is a mechanistic proof of gonadotropin receptor interaction in vivo, which presumably occurs even in the human granulosa cells. In the ovary, LH-like signals could be induced via FSH binding to FSHR during the window of receptor coexpression232. However, results concerning the existence of FSHR–LHCGR transactivation were also provided, although stably transfected HEK293 cells used in this study were not a physiological model237. This issue merits further studies, and alternative experimental approaches may be essential in demonstrating gonadotropin receptor heteromers and complementarity in a physiological context. For instance, bivalent ligands able to bind receptor dimers or super-resolution imaging analysis could be extremely helpful238.Several GPCRs are known to form homo- and hetero-meric functional units consisting of the molecular association between the same or different but structurally similar receptors239, such as those occurring after menopause. Lhr expression was found in the mouse adrenal cortex240, reflecting previous data demonstrating the presence of LHCGR expression in adrenal glands of postmenopausal women241. Aberrant LHCGR expression in the adrenal gland would be linked to cortisol synthesis and Cushing’s syndrome243. Functional LH receptors were also found in uterine tissues of human244 and farm animal models under physiological conditions246. Surprisingly, immunoreactivity to LHCGR was found even in human brain microglial cells and was positively correlated to an increased risk of developing Alzheimer’s disease247. More recently, the presence of LHCGR was suggested in the retina, where LH and hCG would have vascular endothelial growth factor–like functions248.By definition, gonadotropins act in the gonads by binding to receptors specifically expressed therein as a requisite for maintaining endocrine signal specificity and proper metabolic functions. This classic view was recently challenged by several studies describing the expression of functional ectopic gonadotropin receptors, mainly FSHR, supposed to play a significant role in non-reproductive processes. These effects include bone loss, obesity, and cardiovascular and cancer risk, which could be triggered by extragonadal FSHR activated by high FSH levelsCYP19A1 expression and estrogen production249. This is not the only study describing ectopic expression of this receptor; in this regard, an increasing amount of data from human and animal models has been published in the last two decades. Genetically modified or antibody-treated mouse models elegantly suggested that FSH-induced signals modulate bone mass and adipose tissue250. Around these issues, several studies described a link between FSH, osteoporosis, and increased fat mass that occurs after menopause258. Similar conclusions were achieved by in vitro studies using human cells260, although the existence of direct causality between gonadotropin receptor and the physiological effect was questioned261. Relatively low levels of FSH were associated with increased cardiometabolic risk and cardiovascular diseases in the Chinese population262, and the rationale of this issue is provided by the angiogenetic support exerted by FSH via the PI3K/AKT pathway263. Finally, it was demonstrated that FSHR is expressed in tumor vessel cells upregulated by FSH266. These accumulating experimental data seem to clearly debunk the classic view of gonadotropin physiology, potentially leading to new diagnostic, therapeutic, and prognostic opportunities. For instance, the use of anti-FSH antibodies for bone- and fat-specific targeting was proposed for clinical testing267. However, the issue is highly debated, and several objections, including the low reliability of the antibodies used for investigating FSHRs in non-canonical tissues268, firmly oppose the existence of extragonadal FSHRs and LHCGRs269. These data would rely on the discrepancy between mRNA and protein level detected in the analyzed samples, suggesting that they could be an artifact due to suboptimal antibody binding270 and challenging Western blotting271. Missing control experiments of target mRNAs by DNA sequencing263 put further concerns on the reliability of the detection. Together with the aforementioned experimental issues, the opposite results were also found. For instance, a decrease in body fat was not found in mice and humans maintained under gonadotropin production blockade by GnRH agonist/antagonist274. Finally, studies revisiting previous experimental conditions used for detecting extragonadal FSHRs in the human umbilical vein endothelial cells failed in replicating results275, and several technical concerns arose when ectopic gonadotropin receptors were evaluated in other tissues269. In conclusion, the concept of extragonadal FSHRs remains highly controversial. Genetically modified mice may not be the best tools for assessing this issue. Interventional studies in humans will be the only way to demonstrate direct causality between gonadotropin receptors and the abovementioned effects.Functional FSHRs were found in endometriotic lesions, where FSH may induce in vivo, and the role of extragonadal receptors warrant further research. Studying these aspects is vital for further scientific and clinical progress that could benefit human reproductive health.Significant steps forward in the elucidation of gonadotropin signaling and regulation have been made in recent years. The current knowledge addressed most of the questions of the past, revealing novel aspects of FSH, LH, and hCG physiology and providing the potential for new therapeutic applications. Despite these advances, many unanswered questions remain, and the full potential of gonadotropin action, the presence of FSHR–LHCGR heteromers


Insights into the cellular activity of each member of the neurovascular unit (NVU) is critical for understanding their contributions to neurovascular coupling (NVC)—one of the key control mechanisms in cerebral blood flow regulation. Advances in imaging and genetic tools have enhanced our ability to observe, manipulate and understand the cellular activity of NVU components, namely neurons, astrocytes, microglia, endothelial cells, vascular smooth muscle cells, and pericytes. However, there are still many unresolved questions. Since astrocytes are considered electrically unexcitable, To highlight currently available genetic and imaging tools for studying the NVU—and thus NVC—with a focus on astrocyte We draw attention to some outstanding questions regarding the mechanistic basis of NVC and emphasize the role of astrocytic We provide an overview of the current state of NVC research, focusing on the role of astrocytic Advances in imaging techniques together with improvements in genetic tools have significantly contributed to our understanding of NVC. Many pieces of the puzzle have been revealed, but many more remain to be discovered. Ultimately, optimizing NVC research will require a concerted effort to improve imaging techniques, available genetic tools, and analytical software. This process involves multiple cell types, including neurons, glia , and vascular cells , which collectively form the neurovascular unit (NVU). Members of the NVU collectively act in an integrative fashion to respond to increased neuronal activity by increasing local brain blood flow, a process termed functional hyperemia.,in vivo. We also discuss the utility and limitations of these tools in ongoing NVC research.It has been proposed that, under normal physiological conditions, increases in neuronal activity trigger the release of synaptic glutamate, which subsequently activates two different signaling pathways that ultimately elicit functional hyperemia: neuronal-dependent signaling pathways, and astrocyte-dependent signaling pathways.2–in vivo multiphoton fluorescence imaging has enhanced our ability to examine the interaction of members of the NVU without the confounding effects of anesthesia.–NVC forms the basis of functional brain imaging techniques such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), functional magnetic resonance imaging (fMRI), and functional near-infrared spectroscopy (fNIRS), which continue to play important roles in the study of hemodynamics. But how the signals captured by these techniques relate to neuronal activity remains incompletely understood. Accordingly, a rigorous understanding of the interactions between members of the NVU at micrometer-to-millimeter scales of vascular and neural reactivity is critical for more accurate interpretations of functional imaging data. New imaging techniques, in combination with the development of various transgenic mouse models, have significantly advanced our understanding of NVC at the cellular level.2.1–d-aspartate (NMDA) and ,,in vivo data.,,,,–,Inputs from thalamocortical glutamatergic afferents to the neocortex are critical for initiating NVC and subsequent functional hyperemia.2.2,42Astrocytes, the most abundant cell type in the central nervous system (CNS), exhibit a tiled distribution, occupying largely nonoverlapping territories such that only the farthest reaches of their processes come into close proximity with each other. Astrocytes interact with multiple cell types in addition to other glial cells, including neurons, and vascular cells, and release numerous substances upon activation, many of which have functions that remain unclear.ex vivo brain slices has further shown that elevations in astrocytic endfoot ,Despite this general appreciation, the mechanistic details of NVC, including the contribution of astrocytic in vivo studies, however, has introduced some uncertainties regarding the role of astrocytic ,,,,Evidence from 3ex vivo or in vivo. Intracellular ,Since the first report of a synthetic In vivo studies in which astrocytic ,,The first direct evidence in support of a role for astrocytic Although synthetic Aequorea victoria.in vivo studies where it is necessary to resolve motion artifacts or variations in sensor expression among cell populations in a tissue.,–in vivo could partly reflect the contribution of different ,,,The first protein-based ,Glt1, Gfap, Glast, Aldh1l1, andS100β promoters, have been developed and optimized. For induction in adult mice, the tetracycline-inducible Aldh1l1-Cre/ERT2 mouse line is currently the best mouse line option, offering better temporal control of gene expression, high astrocyte specificity, and efficiency.,Aldh1l1-Cre/ERT2 in combination with cyto-GCaMP6f or Lck-GCaMP6f, whether delivered via viral vectors  or transgenic mouse lines, for studying distinct aspects of ,The powerful impact of GECIs on the study of 4Imaging techniques such as intrinsic signal optical imaging (ISOI), laser speckle imaging,in vivo imaging of vascular responses concomitantly with neural ,––,,,–In parallel with the development of GECIs, the introduction of TPLSM,–TPLSM technology is rapidly developing, with new TPLSM modules offering a number of advanced features for the study of NVC  lens allows optical access to subcortical and deep brain regions and enables ,in vivo volumetric imaging to a depth of The recently reported heterogeneous nature of astrocytic ,,Volumetric imaging with a Bessel beam makes it possible to simultaneously image 5,,,,Optical imaging and genetics tools have made significant contributions to research on NVC mechanisms, significantly advancing our understanding of NVC and its essential role in CBF regulation. Much remains to be uncovered; in particular, the underlying mechanisms of NVC and the contributions of neurons and astrocytes to the initiation and maintenance of functional hyperemia remain to be verified. Whether direct targeting of neuronal information to the vasculature takes precedence over the relay of neuronal information from astrocytes to the vasculature, or vice versa, under certain conditions remains an open question. Also unknown is whether both signaling pathways work concomitantly, and if so, under what scenarios might this occur and how might it be advantageous. New evidence continues to provide fresh insights into previously conflicting findings while opening other avenues for examining NVC. Advances in optical imaging in combination with optogenetics and/or pharmacogenetics for monitoring and/or manipulating cellular activity in specific cell types will allow researchers to better interrogate the involvement of individual cells of the NVU in the process. For example, more effort is still needed to elucidate the fascinating and complex variety of astrocytic


As the largest vital solid organ in the body, liver is consisting of multiple types of cells including hepatocytes, Kupffer cell, hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), and other immune cells. The communication between these cells is critical in maintaining liver function homeostasis, and dysregulation of such communication contributes to the pathogenesis of various liver diseases. Extracellular vesicles (EVs), including exosomes and ectosomes, act as important mediators of cell-to-cell communication. EVs can be produced and uptaken by a wide range of cells including all types of cells in the liver. Growing evidences show that EVs are involved in the development of liver diseases, especially non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD). In this review, we will summarize recent advance in how EVs production are altered in NAFLD and ALD and how the changes of EVs quantity and cargos influence the progression of these diseases. The therapeutic and diagnostic potential of EVs in NAFLD and ALD will be also discussed in this review. The term extracellular vesicles (EVs) refers to a highly heterogeneous population of cell-derived membrane-enclosed structures . EVs canIn the field of liver diseases, the number of publications about EVs has grown exponentially over past decade. EVs are emerging as key players in the pathogenesis and progression of metabolic liver diseases including non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease ALD; . NAFLD aHere, we focus on the current state of knowledge pertaining to EVs, including how EVs production is regulated in NAFLD and ALD and how EVs mediated intercellular communications contribute to the pathogenesis and progression of NAFLD/ALD. We will also discuss possible therapeutic and diagnostic applications of EVs in NAFLD and ALD.in vitro and in vivo. For example, all types of cells in the liver have been proved to generate EVs under in vitro conditions , Kupffer cells, liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and other immune cells . In bothzed mice . These sin vivo NASH model further confirmed the critical role of ER stress in controlling EVs release from liver. High fat, fructose, and cholesterol diet activated IRE1α signaling and EVs generation, while mice with liver specific knockout of IRE1α blocked the elevation of EVs in this model  induced toxicity in hepatocytes . AnotherAll these studies have established solid evidences for the key role of EVs in the development of ALD. We have summarized these mechanisms in via S1P1 receptor mediated macrophage chemotaxis and enhanced liver inflammation  and integrin ανβ-5 expression and decreased matrix metalloproteinase (MMP)-7 and plasminogen activator inhibitor-1 expression in hepatocytes, while increased expression of TIMP-1, TIMP-4, Smad-3, integrin ανβ-5, integrin ανβ-8, and MMP-9 in HSCs. These changes caused dysregulation of TGFβ signaling pathway and may contribute to liver fibrosis in NASH  and consequent phenotypical switch from quiescent to activated HSCs . The anglization .via transfer of sphingosine kinase 1 (SK1) and its product S1P , which may cause enhancement or fine tuning of fibrogenic signaling in response to chronic liver injury . Endotheduct S1P .Taken together, EVs released from stressed/injured hepatocytes target liver non-parenchymal cells such as macrophages and HSCs, linking lipotoxicity with events such as inflammation and fibrosis, which are associated with the progression of NAFLD to NASH. In addition, hepatic non-parenchymal cell-derived EVs are also contributed to the development of NAFLD. We have summarized the EVs in NASH in From the perspective of disease treatment, unmodified or engineered EVs can be used as therapeutics for liver diseases. The therapeutic potential of EVs has become a hot research topic recently . Due to Most of engineered EVs use different methods to load the required goods such as specific biomolecules and small molecule drugs into EVs for targeted therapy . In termExtracellular vesicles have broad prospects in the therapeutic field. They have the ability to overcome natural obstacles, their inherent cell targeting properties, and their stability in the circulation . HoweverAs mentioned above, the total number of circulating EVs was significantly increased in patients with various liver diseases. Based on the stability of EVs, it exists in most body fluids and iconic cargoes such as nucleic acid and protein, so it has the potential to be a circulating biomarker . NumerouIn recent years, EVs in the field of metabolic liver disease has gathered significant traction. During lipotoxicity and alcohol exposure, EVs are released in large quantities through different mechanisms. These EVs plays a key role in the transmission of information between different cell types in the liver. They can change the function of target cells and activate different pathways, thereby promoting the occurrence, pathogenesis and development of NAFLD and ALD. Understanding these issues will help us to understand the mechanism of disease and study potential therapeutic interventions. EVs may provide new clues for the therapeutic and diagnosis, but this field is still in its infancy. With more research on EVs, it is believed that EVs can be successfully used in the treatment and clinical diagnosis of NAFLD/ALD in the near future.DW, HZ, and HW contributed to conception and design of the work. DW wrote the first draft of the manuscript. HZ and HW wrote the sections of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With an approach combining crop modelling and biotechnology to assess the performance of three durum wheat cultivars  in a climate change context, weather and agronomic datasets over the period 1973–2004 from two sites, Benatzu and Ussana , were used and the model responses were interpreted considering the role of DREB genes in the genotype performance with a focus on drought conditions. The CERES-Wheat crop model was calibrated and validated for grain yield, earliness and kernel weight. Forty-eight synthetic scenarios were used: 6 scenarios with increasing maximum air temperature; 6 scenarios with decreasing rainfall; 36 scenarios combining increasing temperature and decreasing rainfall. The simulated effects on yields, anthesis and kernel weights resulted in yield reduction, increasing kernel weight, and shortened growth duration in both sites. Creso (late cultivar) was the most sensitive to simulated climate conditions. Simeto and Duilio (early cultivars) showed lower simulated yield reductions and a larger anticipation of anthesis date. Observed data showed the same responses for the three cultivars in both sites. The CERES-Wheat model proved to be effective in representing reality and can be used in crop breeding programs with a molecular approach aiming at developing molecular markers for the resistance to drought stress. SSP1-1.9 represents the low end of future emissions pathways. At the opposite end of the range, SSP5-8.5 represents the very high warming end of future emissions pathways from the literature. SSP2-4.5 represents a scenario with stronger climate change mitigation and lower GHG emissions” ) is the most widely grown crop, accounting for half of the total world production ..73].DREB2 genes and related to drought. The transcript isolated in barley derives from an alternative splicing of a gene, named HvDRF1, where Hv stands for Hordeum vulgare, generating three transcripts. The primer pair used for these experiments was compatible with these transcripts and produced fragments at about 580 bp, 500 bp and 450 bp, which is the same pattern observed in durum wheat. This result led to the conclusion that in durum wheat a homologous gene to HvDRF1 is present and it was named Triticum durum Dehydration-Responsive Factor 1 (TdDRF1). Further studies revealed that this gene produces three transcripts by alternative splicing: TdDRF1.1, consisting of four exons, from E1 to E4; TdDRF1.2, consisting of three exons E1, E2 and E4; and TdDRF1.3, consisting of two exons E1 and E4 [TdDRF1.2 transcript was always expressed at higher levels, the TdDRF1.1 transcript was the least expressed and the TdDRF1.3 transcript was intermediate between TdDRF1.2 and TdDRF1.1 transcripts. These results suggest a correlation between water stress and the expression profile of the TdDRF1 gene and its transcripts.Moreover, sequencing and aligning with TC85717 500 bp bands from all these cultivars, the presence of a short insert 53-bp was detected, revealing a complete homology with transcripts found in barley, homologous to 1 and E4 . This geThe analysis of the historical weather data set covering the study area over the period 1974–2004 confirmed an overall trend with increasing temperatures and decreasing and/or more erratic precipitations. Mean temperatures A showed Calibration and validation of the CERES-Wheat crop model in the study area was already discussed in , where fThe model proved to be less satisfactory in the case of kernel weight. This was probably due to modelling inaccuracies in simulating underlying physiological processes under stressed and non-stressed conditions . Interes2 effect that was not taken into consideration in this study, limiting evaluation of the cultivar responses to climate stimuli.The negative effect of increasing temperatures and decreasing rainfall on simulated grain yields at the two experimental sites is clear . InteresConcerning grain quality, the slight positive effect of increasing temperature and decreasing rainfall on kernel weight  is in coAs for phenology, the shortening of the growing period highlighted in this study is in agreement with other studies ,37,92. IRegarding cultivar choice, this study confirms its potential key role as a farm-level adaptation measure to reduce the negative impacts of climate change on crop production . In partDREB genes in abiotic stress conditions as shown by Liu et al., in 1998 [TdDRF1) has been confirmed both in greenhouse and in field conditions in other durum wheat and triticale cultivars [TdDRF1 gene has been demonstrated in recent studies [TdDRF1.3 transcript in some drought tolerant and rustic durum wheat and triticale cultivars was found [TdDRF1 gene expression as well as the contribution of other genes.A last crucial issue is related to molecular responses in climate change conditions. Since global warming, heatwaves, droughts and a general decreasing rainfall trend are projected in the Mediterranean areas , identif in 1998 . Furtherultivars . Moreove studies  and a coas found . HoweverThis study highlights the importance of a multidisciplinary approach involving the use of crop modelling and biotechnology in order to predict and evaluate the performances of durum wheat genotypes under climate change conditions. Concerning crop modelling, CERES-Wheat proved to be an effective tool when used to predict grain yield, anthesis date and, to a lesser extent, kernel weight. The impact of climate change scenarios on grain yield is more negative, moving from mild to severe scenarios for all genotypes, but reductions are to some extent mitigated for Simeto and namely Duilio (early genotypes) in comparison with Creso (late genotype). On the other hand, kernel weight tends to increase slightly in response to increasing temperatures and decreasing rainfall in particular under mild climate change scenarios. All genotypes showed a reduction of their crop growing cycle as a consequence of increasing temperatures, with Duilio and Simeto revealing to be more resilient than Creso. The detrimental joint effect of simulated increasing temperatures and decreasing rainfall is also affected by soil fertility, with a stronger impact in low-yielding potential soils.DREB genes, with a special focus on TdDRF1, in conditioning the resistance of genotypes to drought conditions must be underlined.The predictive responses of the CERES-Wheat model can be also interpreted in the light of molecular responses of durum wheat cultivar to drought stress. In this context, the role of Furthermore, our analysis indicates that CERES-Wheat crop model responses are highly consistent with observations from most rain-fed durum wheat growing areas of the Mediterranean Region. By showing that early genotypes can be better adapted to increasing temperature and decreasing rainfall, the CERES-Wheat model proves to be a reliable tool to determine the impact of climate change on crops and can help to underline and quantify the simulated effects of cultivar choice to tackle downward trends in grain yield, particularly in the Mediterranean rain-fed areas. Hence, CERES-Wheat can be successfully used to support adaptation strategies such as targeting cultivars onto specific environments or to guide selection decisions in crop breeding programs, also implying the contribution of a molecular approach aiming at developing molecular markers for the resistance to abiotic and drought stresses.


Magnaporthe oryzae, causative agent of the rice blast disease, is a major concern for the loss in yield of rice crop across the globe. It is known for its characteristic melanised dome-shaped appressorium containing a dense melanin layer. The melanised layer is of considerable importance as it is required to generate turgor pressure for initiating peg formation, consequently rupturing the plant cuticle. Various virulence factors play an important role in the disease progression as well as pathogenesis of the fungus. Some of the proteins encoded by virulence genes are associated with signalling, secondary metabolism, protein deprivation, defence responses and conidiation. The purpose of this review is to describe various fungal virulence determinants and provide insights into the molecular mechanisms that are involved in progression of the disease. Besides, the recent molecular approaches being employed to combat the rice blast have also been elaborated. Oryza sativa) is an essential food crop consumed by over 3.5 billion people across the world  in structures called asci , the first stable intermediate. 4THN is reduced to scytalone by 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR), then dehydrated to 1,3,8-trihydroxynapthalene (3THN) by the scytalone dehydratase (SD). The 3THN is further reduced to vermelone by 1,3,8-trihydroxynapthalene reductase (3HNR) and dehydrated by SD to yield 1,8-dihydroxynaphthalene (DHN)  to the maturing appressorium . In absence of cAMP, conidia produce long germ tubes without tip differentiation on the hydrophilic surfaces. Molecular studies have confirmed the role of cAMP signalling in surface recognition and initiation of appressorium formation  kinase  genes, including several Pth11-like genes with the CEFM domain. Zhang et al.  of invading pathogen and elicits PAMP-triggered immune response  is of prime importance due to its role in a number of blast R genes involved in rice breeding  Major resistance (R) genes that confer race-specific resistance and b) quantitative trait loci (QTLs) that control partial, nonrace-specific resistance  is maintained by superoxide dismutase2 targeted by miR398. MicroRNA miR169 acted as a negative regulator in rice immunity against M. oryzae by repressing the expression of nuclear factor Y-A genes  and microRNA (miRNA) are expressed by plants under biotic stress conditions  of sodium hypochlorite were successful in reducing blast infection. Moreover, sodium hypochlorite concentrations and soak time required to eliminate Azoxystrobin is the most commonly used rice fungicide in the southern United States and Asian countries Groth . InformaIprobenphos (IBP) has been popularly used for controlling blast disease of rice along with Edifenphos (EDDP) after phosphorothiolate (PTL) compounds were discovered to exhibit fungicidal properties. IBP is a systemic fungicide, and EDDP is non-systemic fungicide showing effective fungicidal activity when used as a foliar applicant against the blast causing fungus  accumulation in the cell. Increased concentrations of tricyclazole followed by pyroquilon induce flaviolin accumulation, signifying that alternative step of inhibition is present in the pathway. The chemical also obstructs secondary infection by decreasing the sporulation of M. oryzae under field settings. Field studies indicated long-lasting inhibitory potential of tricyclazole and pyroquilon . Other systemic fungicides that function as inhibitors to melanin biosynthesis are pyroquilone, diclocymet and fenoxanil (Nishimura and Hino Magnaporthe isolates by studying major blast resistance (R) gene. This approach is utilised for designing better resistance breeding strategies. In this context, allele mining for resistance genes in all sequenced rice genomes shows the presence/absence of polymorphism and a large number of structural variations  are one of the vital regulators for development and defence in eukaryotic species. miRNAs are implicated in providing immunity against spread of pathogenic organisms in plants, which could be used as biological control agents  and application of fungicides in the fields (to prevent infection of leaves and panicles during the growing season). These techniques have been used to manage and reduce rice blast, but neither one is considered to be highly successful. Recent advances in molecular techniques have been used as an effective tool to understand the biological pathways as well as genes taking part in host-pathogen infection pattern, plant response and disease development.Rice blast, caused by M. oryzae could help to prevent the onset of blast disease, limiting the use of harmful chemicals as pesticides. Recent practices like mutation breeding, MAS for blast resistance in rice programme, miRNA in blast disease management, transgenic approaches for blast resistance can also play key role in monitoring blast disease.The understanding of major molecular targets associated with cell wall, signal transduction pathways and host–pathogen interactions in


Dictyostelium discoideum aggregate and eventually form fruiting bodies consisting of stalk cells and spores. We focus on how this bifurcation of cell fate is achieved. During growth mTORC1 is highly active and AMPK relatively inactive. Upon starvation, AMPK is activated and mTORC1 inhibited; cell division is arrested and autophagy induced. After aggregation, a minority of the cells  continue to express much the same set of developmental genes as during aggregation, but the majority (prespore cells) switch to the prespore program. We describe evidence suggesting that overexpressing AMPK increases the proportion of prestalk cells, as does inhibiting mTORC1. Furthermore, stimulating the acidification of intracellular acidic compartments likewise increases the proportion of prestalk cells, while inhibiting acidification favors the spore pathway. We conclude that the choice between the prestalk and the prespore pathways of cell differentiation may depend on the relative strength of the activities of AMPK and mTORC1, and that these may be controlled by the acidity of intracellular acidic compartments/lysosomes (pHv), cells with low pHv compartments having high AMPK activity/low mTORC1 activity, and those with high pHv compartments having high mTORC1/low AMPK activity. Increased insight into the regulation and downstream consequences of this switch should increase our understanding of its potential role in human diseases, and indicate possible therapeutic interventions.mTORC1 and AMPK are mutually antagonistic sensors of nutrient and energy status that have been implicated in many human diseases including cancer, Alzheimer’s disease, obesity and type 2 diabetes. Starved cells of the social amoeba  D. discoideum aggregate to form loose mounds, which are transformed into “tight aggregates”, in which the amoebae are closely associated via lateral and polar contacts and become surrounded by a complex extracellular matrix of protein and cellulose to create, in effect, a multicellular organism  and AMPK (AMP-activated protein kinase), and on the fact that and AMPK  have emeon in G1  and the on in G1 .D. discoideum are starved: mTORC1 activity is rapidly down-regulated and AMPK activity increases  to IMP (inosine 5-monophosphate) and as a result accumulate abnormally high levels of AMP . These s of AMPK . Similar of AMPK . However of AMPK , supportin vivo as we propose, we would expect it also to play a role in DIF-induced stalk cell formation. In agreement with this suggestion, exposure of Dictyostelium amoebae to DIF-1 leads to extensive changes in protein phosphorylation at sites many of which fit the AMPK consensus motif  [see ]. A provcAMP receptor → Zak1 → GskA--→ Aar (a β-catenin homolog)→ psp gene expression.For reasons that are not understood, mutations affecting this pathway have very variable effects on the extent of prespore and spore cell formation [see for example ]. On theDictyostelium mutant has been described in which the gene encoding 1-phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) has been disrupted (2 (PIP2), and there is strong evidence that PIP2 is located on the vacuole membrane of yeast and on acidic vesicle/endosomal membranes in mammalian cells and act as an anchor to associate mTORC1 to these membranes .Until recently there was no evidence connecting the spore pathway with mTORC1. Any effect of mTORC1 could involve a parallel pathway or mTORC1 could influence the activity of the above pathway. There is clearly a complex interaction between mTORC1 and Gsk3 activity in humans which is not fully understood  and has isrupted , and proembranes , a precoDictyostelium will have a bearing on the development of such inhibitor treatments.In humans, the central roles of PIKfyve in endosomal trafficking and autophagy make it an attractive candidate for treatment of a number of diseases, using PIKfyve inhibitors such as apilimod. These diseases include cancer and neurodegenerative conditions as well as viral infections including those due to coronaviruses . Understin vitro to weak bases switches them from the stalk to the spore pathway, whereas weak acids have the opposite effect ] are highly acidic and are stained by the weak base, neutral red, whereas at the first finger and slug stage of development, the AVs of prespore cells have lost their acidity and are unstained . We sugge effect  while ate effect . Althouge effect . This efIn contrast to the marked difference between prestalk and prespore cells in terms of pHv, their cytosolic pH values are either not different  or diffeFurther evidence that pHv influences cell fate choice comes from the remarkable 1-dimensional differentiation system devised by Bonner . In this2  . Autopha pathway ; mutatioDictyostelium has implications for treatment of a range of human diseases. For example, ketogenic diets, which involve intake of high levels of fat and low levels of carbohydrate, were developed to mimic starvation , a major component of cannabis oil, enhances mTORC1 activity in Dictyostelium in a mechanism dependent on inositol polyphosphate multikinase (Dictyostelium identifies CBD as a potential therapy for multiple sclerosis through increased mTORC1 activity. DIF-1 inhibits the growth of a range of different cancer cells (Dictyostelium. It will be of interest to define the role of the pH of acidic vesicles in the effect of DIF-1 on growth of these cancer cells as alteration of this may offer an alternative therapy route.Understanding the mechanisms and consequences of mTORC1 regulation in arvation . Such diarvation . Studiesignaling . This wotikinase . This paer cells . The mecer cells  in a patDictyostelium may provide a tractable non-metazoan model for examining in detail how the two ancient sensor molecules, mTORC1 and AMPK, control the differential gene expression underlying a cell fate choice.In conclusion,