{"paper_id":"14ff9924-6321-4b14-b6de-2671a87c0819","body_text":"The RhoA is a small GTPase that was first discovered in the mollusk  Aplysia  and then in humans in 1985 by Madaule and Axel [ 1 ] . RhoA functions by cycling between an active GTP-bound state and an inactive GDP-bound state, changing its conformational state and relative activity. The GTPase activity of RhoA is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), with further regulatory effects by guanine nucleotide-dissociation inhibitors (GDIs) [ 2 ] . GAPs inactivate RhoA by increasing the rate of GTP hydrolysis, returning RhoA to its inactive form, whereas GEFs provide a counter-mechanism by expediting the release of GDP, thereby allowing new GTP to bind RhoA. Specific GAPs and GEFs can be turned on or off by various molecular signals, allowing for finely tuned control of RhoA activity.\nRhoA activity is associated with its cellular localization. For example, p115RhoGEF binds to active G protein-coupled receptors (GPCRs) in the plasma membrane and activates RhoA upon ligand binding [ 3 ] . Common GPCR ligands for RhoA activation include thrombin, lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), and thromboxane A2 [ 4 , 5 ] . When bound to GDP, RhoA is sequestered in the cytoplasm by RhoGDIs in its latent state. RhoA is fully activated upon translocation to the membrane and dissociation from the GDI. Dissociation from GDI may be facilitated by a GDI displacement factor (GDF), which binds to RhoGDI to facilitate RhoA release and activation [ 6 ] , or by phosphorylation events initiated by kinases, such as protein kinase C (PKC) or p21-activated kinase (PAK) [ 7 ] . After activation, RhoA is either located in the cytosol or associated with the plasma membrane  via  its prenyl group [ 3 ] . Although GTP-bound RhoA is assumed to be active, an inactive conformation of GTP-bound RhoA has been reported [ 8 ] . In addition, GTP-bound RhoA may be localized in the cytosol or at the membrane; however, membrane-localized GTP-bound RhoA is required for the activation of RhoA downstream signaling [ 9 ]  (Fig.  1 ). Further, RhoA can localize within the nucleus for actin polymerization and DNA damage response, with the involvement of various GEFs and GAPs present within the nucleus [ 10 ] .\nIntracellular RhoA signaling. RhoA may be activated  via  GPCR-induced G protein activation of RhoGEF, which exchanges GDP for GTP to activate RhoA. Active RhoA may interact with a variety of downstream effectors, including ROCK1/2. RhoA is inactivated by RhoGAP, which accelerates the hydrolysis of GTP to GDP. Inactive RhoA is sequestered by RhoGDI, which forms a complex with GDP-RhoA. Phosphorylation events initiated by kinases such as PKC or PAK phosphorylate sites on RhoGDI or actions of RhoGDF initiate dissociation and free GDP-RhoA to continue the cycle. GPCR: G protein-coupled receptor; PAK: p12-activated kinase; PKC: protein kinase C; RhoA: Ras homolog gene family member A; RhoGAP: Rho GTPase activating protein; RhoGDI: Rho guanine nucleotide-dissociation inhibitors; RhoGEF: Rho guanine nucleotide exchange factors; ROCK1/2: Rho-associated coiled-coil kinases 1/2. The figure was generated using BioRender.\nRhoA activity is also regulated at transcriptional and post-translational levels. The transcription factors Myc, hypoxia inducible factor 1 (HIF-1), signal transducer and activator of transcription 6 (STAT6), and nuclear factor-kappa B (NF-κB) are involved in  RhoA  transcription in various physiological contexts [ 3 ] . Myc may also be involved in a negative feedback mechanism to post-transcriptionally block RhoA/ROCK signaling, which is involved in cytoskeletal function [ 11 ] . RhoA may be modified  via  ubiquitylation of lysine; phosphorylation of tyrosine, threonine, or serine; nitration of tyrosine; and AMPylation of tyrosine at conserved sites. Nitration of tyrosine Y34 can reduce GDP affinity, whereas AMPylation of Y34 can block interactions with downstream effector proteins, thus serving as an additional control mechanism over RhoA activation/inactivation [ 12 ] . Serine/threonine phosphorylation at S/T26 also inhibits RhoA, whereas lysine ubiquitylation is thought to increase protein stability. The tyrosine residues Y34 and Y64 are phosphorylated under normal conditions.\nActive RhoA can activate its downstream proteins Rho-associated coiled-coil kinases 1 and 2 (ROCK1/2, or ROK, Rho-kinase, and p160ROCK, colloquially referred to as ROCK(s)), p140mDia (mDia), and other targets [ 13 ] . ROCK proteins regulate actin organization, apoptosis, tissue and organ development, cell proliferation, and cytokinesis [ 14 ] . mDia inhibition is relieved by a conformational change upon RhoA binding, enabling mDia to play important roles in actin polymerization and cytokinesis [ 13 ] . ROCK proteins are ubiquitously expressed, with high ROCK-1 expression reported in all tissues except the brain and muscle, whereas ROCK-2 expression is higher in the brain, muscle, heart, lungs, and placenta [ 15 ] .\nRhoA signaling has several functions, including the regulation of cell proliferation, survival, migration, adhesion, cytokinesis, stress fiber formation, apical constriction, stem cell lineage fate, vesicle trafficking, and gene expression [ 4 , 16 – 21 ] . Despite its ubiquitous expression in eukaryotic cells, RhoA has essential tissue- and cell type–specific functions, such as protection from heart injuries, regulation of lens epithelium morphology and the hematolymphoid system, diverse functions in different parts of the nervous system [ 17 ] , progesterone (P4) steroidogenesis in the corpus luteum (CL) [ 22 ] , and regulation of immune responses [ 23 , 24 ] , that have been revealed in conditional RhoA knockout mouse models.\nRhoA signaling can be studied using various methods, both  in vitro  and  in vivo . Classical approaches before the development of live fluorescent probes measured active RhoA activity using an active RhoA-specific antibody, analyzed through pulldown assays and visualized through localized fluorescence [ 25 ] . Modern techniques have since been developed, allowing for live spatiotemporal reporting of active RhoA. The first technique uses a binding domain specific to RhoA-GTP fused to a fluorescent probe with the ability to relocalize to the native location of active Rho GTPase [ 26 ] . Although groundbreaking, this technique has the following disadvantages: competition with endogenous effectors and no preferential binding between RhoA, RhoB, and RhoC. A second method has been developed that uses fluorescence resonance energy transfer (FRET), which relies on exogenous RhoA fused to a RhoA-binding domain that may accomplish FRET upon activation, allowing localized reporting of activated exogenous RhoA [ 27 ] . However, this method has the disadvantage of only measuring exogenous RhoA, which may localize and migrate differently when overexpressed compared with endogenous RhoA. Both approaches are robust for  in vitro  labeling of GTP-bound RhoA. Notably, the approach of using FRET-competent RhoA can also be modified for  in vivo  use by creating a RhoA-FRET mouse model that utilizes a lox-stop-lox site coupled with Cre recombinase for conditional expression in specific tissues [ 28 ] . Other  in vitro  strategies include mutant overexpression of RhoA or inactivation of RhoA, RhoB, and RhoC  via Clostridium botulinum  C3 toxin; however, these approaches may result in the loss of some of the overarching signaling pathways and motifs owing to the use of immortalized cell lines and general  in vitro  conditions, which may alter the biochemical environment [ 17 ] . The cyclic nature of GTPases makes it challenging to study the real-time functions of active RhoA, especially  in vivo . The Cre-loxP system can be used to conditionally delete RhoA from specific cells or tissues.\nElucidating the upstream and downstream signaling regulators and effectors of RhoA is more difficult as there are a variety of GEFs, GAPs, and GDIs that are specific to cell types and have varied cascades to impart the effects of RhoA [ 3 , 4 ] . An informed approach is essential when knocking out one of these proteins to study its function. For example, knockout of the GEF  Ect2  in mice was informed by its interphase nuclear localization and release during cytokinesis, and  Ect2  knockout caused impaired cell proliferation and migration [ 29 ] . This finding is consistent with the known functions of RhoA in cytokinesis. However, some effectors may also display phenotypes atypical to established RhoA functions, such as the RhoGEF  rgnef , the knockout of which in MEF cells blocks focal adhesion formation [ 30 ] , whereas RhoA knockout MEF cells showed no alterations in focal adhesions [ 31 ] . ROCK kinases also present an opportunity to evaluate RhoA signaling, and deletion of either of these kinases or their own downstream targets may be a promising approach to elucidate the latent functions of RhoA. For example, deletion of ROCK-1 or ROCK-2, or mutation of their downstream target, MAP3K1, results in an eyes-open-at-birth phenotype in mice, which is atypical for murine development. Similar results were observed with RhoGEF deletion; however, RhoA deletion did not result in alterations in eyelid development [ 32 , 33 ] . The existing literature illustrates the possibility of multiple pathways that act on RhoA and its regulators and effectors, necessitating an investigation of the GTPase itself and an independent investigation of upstream and downstream targets.\nThe mammalian FRS generally consists of two ovaries, two fallopian tubes/oviducts, a uterine body composed of one central compartment or two uterine horns, a placenta/placentas that transiently develops during pregnancy, a cervix, a vagina, and mammary glands. The functions of the FRS are under the central control of the hypothalamic–pituitary–ovarian (HPO) axis and are regulated by local factors [ 34 – 38 ] . RhoA is expressed in cells of the FRS and RhoA signaling has been noted in FRS tissues/cells [ 39 – 48 ] . These findings have led to a greater understanding of the  in vivo  functions of RhoA. This is the first review of the function of RhoA in the FRS. It is organized as follows: ovary, oviduct, uterus/corpus uterus, cervix, placenta, vagina, and mammary gland. Both physiological functions and pathological implications are discussed (Fig.  2 ).\nSummary of reported functions of RhoA in the female reproductive system. CL: corpus luteum; KO: knockout; P4: progesterone; PCOS: polycystic ovarian syndrome; RhoA: Ras homolog gene family member A; StAR: steroidogenic acute regulatory protein; TEB: terminal end buds. The figure was generated using BioRender.\n\nThe ovary contains follicles at different stages of development, including primordial, primary, secondary, and tertiary/antral stages. The key cell types in a follicle include an oocyte, granulosa cells, theca cells, and immune cells [ 35 ] . The main function of follicles is oocyte maturation. Mature oocytes are surrounded by a group of closely associated granulosa cells, called cumulus cells, which are housed within a mature antral follicle. In response to a pre-ovulatory luteinizing hormone (LH) surge, cumulus cells in the antral follicle undergo cumulus expansion, with the production of extracellular matrix (ECM) components and their deposition into the intercellular space to form a tight complex with the oocyte called the cumulus–oocyte complex (COC). Upon ovulation, the COC is released from the mature follicle [ 47 ] . The ruptured follicle that remains after COC release develops into a CL, with the development of vasculature and differentiation of granulosa and thecal cells into luteal cells; therefore, the majority of cells in the CL are vascular endothelial cells and luteal cells [ 35 , 49 , 50 ] . The cumulus cells in the COC protect the oocyte during ovulation and facilitate sperm access to the oocyte for successful fertilization. The ovary produces the ovarian hormones estrogens, with 17β-estradiol (E2) being the most potent endogenous estrogen, and progesterone (P4). Ovarian estrogen synthesis follows the “two cell two gonadotropin” theory, according to which LH activates LH receptors on theca cells leading to increased production of androgens, which is shuttled to granulosa cells, whereas follicle-stimulating hormone (FSH) activates FSH receptors on granulosa cells promoting the conversion of androgens to estrogens [ 51 ] . Ovarian P4 synthesis occurs mainly in the CL. E2 and P4 are essential for the functions of the oviduct and uterus in supporting preimplantation embryo development and transport, embryo implantation, and post-implantation pregnancy.\nDevelopment of the ovarian follicle is essential for proper ovulation. Follicles mature in the ovary and are surrounded by granulosa and theca cells, which function in response to the gonadotropins LH and FSH. In granulosa cells of galline (chicken) pre-ovulatory follicles,  RhoA  is repressed by the transcription factor forkhead box L2 (FOXL2) [ 52 ] . FOXL2 is essential for the regulation of ovarian follicle granulosa cell development, estradiol signaling, and steroidogenesis. FOXL2 depletion in  Foxl2 lacZ  mutant murine ovaries prevents granulosa cell differentiation and secondary follicle formation [ 53 ] . FOXL2 overexpression increased F-actin depolymerization, increased the disorder of actin filaments, and reduced granulosa cell expansion, whereas FOXL2 silencing had the opposite effect. ChIP-seq analysis indicated that FOXL2 directly inhibits  RhoA  expression in pre-ovulatory granulosa cells. Cells treated with the ROCK inhibitor Y-27632 exhibited a microfilament disorder similar to that observed in both the FOXL2 silenced + Y-27632 treated and FOXL2 overexpression groups, suggesting that FOXL2-mediated granulosa cell expansion occurs  via  the RhoA/ROCK signaling pathway, and dysregulated RhoA activity may present a significant barrier to oocyte release.\nIn both porcine and murine models, RhoA has been shown to play a key role in oocyte maturation by promoting actin assembly, spindle formation, and contractile ring formation. RhoGEF ARHGEF2, a guanine exchange factor and RhoA activator, is upregulated after  in vitro  maturation of oocytes in a porcine model [ 54 , 55 ] . Targeting downstream RhoA signaling with Y-27632 inhibited meiotic maturation in porcine cultured oocytes and led to the failure of germinal vesicle breakdown and first polar body extrusion in murine oocytes [ 56 , 57 ] . The maturation-promoting factor/epithelial cell transforming 2 (ECT2)/RhoA pathway is important for the completion of the first meiosis in mouse oocytes by extrusion of the first polar body [ 58 ] . These studies indicate that both RhoA activation and ROCK transduction are necessary for successful early oocyte development. RhoA signaling also indirectly regulates oocyte function  via  the direct regulation of cumulus cells. Prostaglandin E 2  (PGE 2 ) is a key mediator of gonadotropin-induced ovulation. PGE 2  acts through its receptors, EP1 to EP4 (encoded by  Ptger1  to  Ptger4 , respectively) [ 59 ] . EP2 is expressed in cumulus cells and facilitates fertilization by suppressing C-C motif chemokine signaling–mediated ECM assembly [ 33 ] .  Ptger2 −/−  mice have impaired PG signaling, enhancing chemokine-induced RhoA activation and subsequent actin polymerization in cumulus cells, leading to a cumulus ECM barrier resistant to sperm penetration and thus a reduced fertilization rate [ 47 ] . These findings demonstrate the involvement of RhoA signaling in the regulation of COC integrity for sperm penetration. Thus, an investigation of RhoA in the oocyte environment before fertilization is essential to elucidate further mechanisms.\nRhoA, in tandem with the Rho family GTPase Cdc42, may regulate spindle behavior during early embryogenesis, as shown in murine oocytes treated with a central spindle pathway inhibitor in arrested metaphase II before the induced resumption of the cell cycle without fertilization [ 60 ] . RhoA recruitment to the cortical region and subsequent spindle rotation were inhibited, whereas the use of a dominant-negative Cdc42 form (Cdc42T17N) revealed two phenotypes: either a small polar body 2 extrusion or spindle relocation to the cell center. Inhibition of the downstream kinase, ROCK, produced similar detrimental effects on oocyte maturation. Although this study was performed  in vitro  in unfertilized oocytes, the resumption of metaphase II is a core process of embryogenesis; thus, these observed effects may also implicate Rho/ROCK signaling in embryogenesis.\nThe CL is a transient endocrine gland formed after ovulation and has three main stages during its life span: development, maintenance, and regression/luteolysis. The CL is critical to establish early pregnancy and regulate periodic estrous and menstrual cycles. During early pregnancy, the CL is the main site for P4 production to support early pregnancy events, including embryo implantation [ 61 – 64 ] . Disrupted timing (eg, premature CL regression) and impaired P4 synthesis (ovarian/luteal insufficiency) can lead to early pregnancy loss and infertility [ 61 , 65 , 66 ] . The cytoskeleton is essential for P4 steroidogenesis, as demonstrated in vimentin-deficient ( Vim −/− ) mice,  Vim −/−  luteinized granulosa cells [ 67 , 68 ] , and porcine luteal cells [ 69 ] .\nRhoA signaling plays critical roles in regulating the dynamics of cytoskeletal filaments [ 70 – 72 ]  and cytoskeletal proteins, including some in the RhoA signaling pathway (eg, EZR), which are downregulated during luteolysis [ 73 ] . Notably, Semaphorin-4C (Sema4C), a member of the family of morphogenetic semaphorins and a RhoA activator, is abundantly expressed in the ovarian stroma, follicles, and CL. Inhibition of Sema4C by shRNA injected into the ovarian bursa significantly reduced E2, P4, and testosterone levels in mice, and the impaired steroidogenesis could be rescued by treatment with a downstream kinase ROCK1 agonist [ 74 ] , demonstrating the role of RhoA signaling in steroidogenesis in the ovary.\nOur laboratory demonstrated RhoA upregulation during CL development with high expression in mouse luteal cells during CL maintenance. Furthermore, we showed that RhoA deletion in luteal cells (progesterone receptor [PR]-positive cells) resulted in P4 deficiency and infertility in  RhoA f/f Pgr Cre/+  mice [ 22 ] . The normal number of ovulated oocytes detected on day 0.5 post-coitum (D0.5, mating night as D0), coupled with the reduced number of CLs detected on D3.5 (during CL maintenance) in  RhoA f/f Pgr Cre/+  mice compared with  RhoA f/f  control mice, indicates an essential role of RhoA in the luteal cells for CL development.  RhoA f/f Pgr Cre/+  luteal cells have reduced and disrupted cytoskeleton (indicated by β-actin staining and vimentin staining), indicating a critical role of RhoA in regulating the cytoskeleton in the luteal cells. Because the P4 steroidogenesis substrate cholesterol is enriched in lipid droplets and the cytoskeleton facilitates the interaction of lipid droplets with mitochondria to make cholesterol available for P4 synthesis in the mitochondria, the disrupted cytoskeleton in  RhoA f/f Pgr Cre/+  luteal cells likely contributes to the impaired transport of lipid droplets to mitochondria, resulting in lipid droplet accumulation in the cytoplasm.  RhoA f/f Pgr Cre/+  luteal cells also have reduced density of mitochondria, the site of the first step of steroidogenesis, and reduced expression of steroidogenic acute regulatory protein (StAR), the rate-limiting enzyme in steroidogenesis, all of which contribute to P4 deficiency and infertility in  RhoA f/f Pgr Cre/+  mice [ 22 ] . This study demonstrated the essential  in vivo  functions of RhoA in luteal cells during early pregnancy.\nRhoA f/f Pgr Cre/+  CL also has defective vasculature. Compared to its expression in luteal cells, RhoA does not seem to have a significant expression level in the endothelial cells of D3.5 mouse CL; thus, the defective vasculature in the  RhoA f/f Pgr Cre/+  CL may suggest a paracrine effect of RhoA signaling in luteal cells or a secondary effect from defective luteal cell differentiation and CL development [ 22 ] . Interestingly, cellular communication network factor 1 (CCN1/Cyr61), a secreted protein and an angiogenic inducer, is upregulated by RhoA activation in cultured early-cycle bovine luteal cells [ 75 ] , suggesting that RhoA in luteal cells may indirectly regulate the luteal vasculature  via  a CCN1-mediated paracrine mechanism. Whether CCN1 is a downstream target of RhoA contributing to the defective vasculature in the  RhoA f/f Pgr Cre/+  CL remains to be investigated [ 22 ] . Data from the Human Protein Atlas database indicate that RHOA is expressed in both human ovarian endothelial and ovarian lymphatic endothelial cells [ 76 ] . At D3.5, in the ovary of control mice, the RhoA expression level in the medulla, an area with enriched blood vessels, was lower than that in luteal cells [ 22 ] . The  in vivo  functions of RhoA in ovarian endothelial cells have not yet been systematically investigated.\nOvarian cancer is generally divided into three subtypes: clear cell and endometrioid carcinomas, which are thought to arise from endometriosis of the ovary and abnormal microenvironments; mucinous carcinomas, which are associated with the formation of benign teratomas; and serous carcinomas, which can be further subdivided based on the number of genetic mutations and the rate of growth [ 77 ] . Real-time PCR of 22 clear cell carcinomas and 31 high-grade human serous carcinomas indicated that  RhoA  expression levels increased from the early stage to stages II to IV in clear cell carcinomas and that low  RhoA  expression levels correlated with better treatment efficacy in high-grade serous carcinomas [ 78 ] . In both carcinoma subtypes,  RhoA  upregulation was correlated with greater aggressiveness and a higher degree of invasiveness [ 78 ] . Western blotting has also demonstrated that RhoA upregulation is associated with tumor progression in human ovarian carcinoma [ 79 ] . These observations in human ovarian cancer tissues were also corroborated by loss-of-function  in vitro  and  in vivo  studies, where RhoA knockdown in the human epithelial ovarian cancer cell line HO8910 reduced cell viability, migration, invasion, and adhesion. Furthermore, RhoA knockdown in transplanted HO8910 cells in nude mice suppressed tumor xenograft formation and growth and induced tumor cell apoptosis [ 80 ] . RhoA is also detected in the human fallopian tube epithelium, which may be the origin of some ovarian carcinomas, and its expression is increased in primary ovarian carcinomas [ 81 ] . The critical role of RhoA in ovarian cancer progression makes it a potential therapeutic target for ovarian cancer management.\nPCOS is a common disorder of the ovaries characterized by anovulation, elevated androgen levels, subsequent changes in uterine contractility, and abnormal growths on the ovaries. An evaluation of mRNA and protein levels of RhoA and ROCK in a dehydroepiandrosterone-induced PCOS rat model showed no significant changes, and the GTP-bound active RhoA form was not investigated in the study; however, treatment of the PCOS rat model with the Rho-kinase inhibitors fasudil or Y-27632 inhibited the increased uterine contractions associated with PCOS [ 82 ] , suggesting that increased RhoA activity  via  ROCK signaling may be imparted by an increased activity of RhoA activators, such as RhoGEFs; further studies investigating GTP-bound RhoA and GEF/GAP (RhoA activator/inactivator) activity in a PCOS model should be performed (potential functions of RhoA in uterine contractility are reviewed in section “Functions of RhoA in the uterus (corpus uterus)”).\n\nThe oviduct (in rodents)/fallopian tube (in humans) is the site of fertilization and early embryo development and transport. Our unpublished immunohistochemistry data show RhoA expression on day 3.5 post-coitum (D3.5; embryo implantation is initiated at ~D4.0 in mice) in the mouse oviductal epithelium, and our unpublished microarray data revealed that  RhoA  is among the most abundant genes in the D3.5 mouse oviduct. The Human Protein Atlas designates  RHOA  RNA expression to ciliated and secretory cells, endothelia and lymphatic endothelia, smooth muscle and fibroblasts, as well as T cells, plasma cells, granulocytes, and macrophages within human fallopian tubes [ 76 ] . However, the functions of RhoA in the oviduct/fallopian tube have not been systematically investigated. In a study, Ras-related nuclear protein (RAN), which promotes membrane targeting and stabilization of RhoA, was detected in small extracellular vesicles derived from human fallopian tube epithelial cells [ 83 ] . This observation may implicate RhoA signaling as a mediator of cell–cell communication in the oviduct/fallopian tube, such as during fertilization, early embryo development, and embryo transport. It has been demonstrated that CDC42, one of the three most-studied Rho GTPases, is critical for embryo transport in the mouse oviduct [ 84 ] . The potential  in vivo  functions of RhoA in the oviduct/fallopian tube require further investigation, and we are currently investigating the  in vivo  function of RhoA in the oviduct during early pregnancy using a  RhoA f/f Pgr Cre/+  mouse model [ 22 ] .\n\nThe uterus remains the only site that can carry a fetus full term. It undergoes cyclic changes during the estrous/menstrual cycle and dynamic changes during pregnancy. It comprises the perimetrium, myometrium, and endometrium. The endometrium is further divided into the stromal and epithelial layers. The uterine epithelium includes the luminal epithelium (LE) and glandular epithelium (GE). The LE is the first maternal contact for an embryo during implantation to establish the first embryo–maternal interface during normal pregnancy [ 61 ] . Most normal pregnancy events occur in the uterus; these include preimplantation embryo development, embryo implantation, post-implantation embryo/fetal development, placental development and maintenance, and parturition. The Human Protein Atlas shows  RHOA  RNA expression in ciliated cells, glandular and luminal epithelial cells, endothelia, smooth muscle, and stromal cells, as well as T cells and macrophages within the human endometrium [ 76 ] .\nEmbryo implantation is the process by which competent embryos are implanting into a transiently receptive uterus. This is a mandatory step in mammalian reproduction for subsequent embryonic and fetal development. Embryo implantation requires synchronized readiness of the uterus and embryo, as well as timely dialogue between them. Uterine receptivity is coordinately regulated by the ovarian hormones E2 and P4; however, the process by which the uterus reaches a transiently receptive state for embryo implantation is not well understood. The LE serves as the transient gateway for embryo implantation, and the GE secretes molecules critical for embryo implantation [ 61 ] .\nOur microarray analysis showed  RhoA  as one of the most abundant genes in the peri-implantation uterine LE, the initial site of embryo–maternal interactions, with  RhoA  expression level being more than double that of  Cdc42  and  Rac1 [ 40 ] , the other two most-studied members of the Rho family of GTPases. CDC42 downregulation has been implicated in endometrial stromal cell senescence associated with recurrent implantation failure in women [ 52 ] , whereas uterine deletion of Rac1 in  Rac1 f/f Pgr Cre/+  mice leads to impaired uterine receptivity for embryo implantation and, ultimately, infertility [ 85 ] . These studies demonstrated the essential roles of Rho family GTPases in the uterus during early pregnancy. The expression of RhoA in the LE during peri-implantation coupled with the function of RhoA as a mechanosensor supports the hypothesis about the role of RhoA in the LE in sensing an embryo for implantation. However, the function of uterine RhoA in early pregnancy is yet to be systemically investigated in  RhoA f/f Pgr Cre/+  mice because of P4 deficiency as a confounding factor [ 22 ] . Exogenous P4 treatment rescued embryo implantation but with a significantly reduced number of implantation sites in  RhoA f/f Pgr Cre/+  mice, indicating an  in vivo  function of RhoA in the oviduct and/or uterus to support early pregnancy. Exogenous P4 treatment also promotes uterine fluid absorption in  RhoA f/f Pgr Cre/+  mice [ 86 ] , which likely contributes to the rescue of embryo implantation. Although one study suggested that the reduced number of embryo implantation sites following intrauterine injection of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 was owing to reduced  RhoA  expression (~30% reduction compared with the control) at the implantation site [ 87 ] , this conclusion is questionable because embryo implantation requires synchronized readiness of both the embryos and uterus, and LY294002 has a direct adverse effect on preimplantation embryo development [ 88 ] .\nDecidualization is the differentiation of endometrial stromal cells into specialized secretory decidual cells that provide nutrition and an immunoprivileged matrix for an implanting embryo (an embryo is semi-allogeneic to the biological mother and allogeneic to a surrogate woman). Decidualization is essential for successful embryo implantation and placental development. In mice, decidualization is initiated a few hours after embryo attachment [ 89 ] , whereas in humans, decidualization is independent of embryo implantation and is controlled by the secretory phase of the menstrual cycle driven by the postovulatory rise in progesterone levels and increasing local cyclic AMP (cAMP) production [ 90 ] . Impaired decidualization leads to impaired embryo implantation and is a major cause of female infertility [ 90 ] .\nConsidering the dynamic remodeling of the uterus during pregnancy, the functions of RhoA signaling in the cytoskeleton and mechanosensation, and the high levels and widespread expression of RhoA in different uterine cells, RhoA is expected to have  in vivo  functions in the uterus under physiological and/or pathological conditions. Because of the cyclic nature of RhoA activity, which is also regulated by the cellular localization of RhoA, steady-state mRNA or protein expression of total RhoA is inadequate to confer RhoA cellular activity. Approaches using fluorescent probes or FRET could distinguish the spatiotemporal localization of active RhoA in the dynamically remodeled uterus during pregnancy to assist in the investigation of the  in vivo  functions of uterine RhoA in pregnancy.\nIn vitro  studies have implicated Rho/ROCK signaling in stromal/decidual cells and the outgrowth of embryos co-cultured with stromal/decidual cells (as an  in vitro  model for human implantation), albeit with inconsistent results. Spontaneous decidualization in the human endometrium begins in the mid-secretory phase. One study revealed RhoA localization mainly in the GE and stromal cells of human endometrial tissues in the proliferative and mid-secretory phases of the menstrual cycle, and in the GE and decidual stromal cells of human decidua from first-trimester abortion [ 42 ] . In cultured human decidual cells, RhoA exhibits a floccular staining pattern throughout the cytoplasm, with a small portion located at the plasma membrane. Treatment with exoenzyme C3, an inhibitor of RhoA, RhoB, and RhoC proteins, causes cultured decidual cells to round up and lose their actin stress fibers and also decreases the area of trophoblast outgrowth of co-cultured hatched mouse blastocysts, whereas treatment with LPA, an activator of the Rho/ROCK pathway, induces new stress fibers in cultured decidual cells and increases the outgrowth of co-cultured hatched mouse blastocysts on decidual cells [ 42 ] . These observations demonstrate the functions of Rho/ROCK signaling in regulating actin stress fibers in decidual cells and outgrowth of co-cultured embryos. Whether the effects of C3 and LPA on the outgrowth of co-cultured embryos are mediated by decidual cells and/or co-cultured embryos remains to be investigated. In an  in vitro  human endometrial culture, RhoA was identified as a downstream target of annexin A2 (ANXA2), a calcium-binding protein essential for membrane–cytoskeletal interactions, and failure of ANXA2 to activate RhoA results in defective trophoblast outgrowth and adhesiveness [ 41 ] . Similarly, inactivation of RhoA  via Clostridium difficile  toxin A results in the downregulation of trophoblast adhesion in RL95-2 (human epithelial-like cell line derived from the endometrium) cells and JAR (human choriocarcinoma derived from placental trophoblastic cells) cells  in vitro [ 91 ] . These studies using pharmacological approaches indicate an integral role for RhoA signaling in the interactions between co-cultured stromal/decidual cells and trophoblasts. However, another study employed a more specific silencing approach using RhoA RNAi in primary human endometrial stromal cells, and the results suggested an inhibitory role of RhoA signaling on embryo spreading and invasion, which was corroborated in a study using the ROCK inhibitor Y-27632 [ 92 ] . This study also showed that Rac1, one of the three most-studied members of the Rho family of GTPases, has opposite effects on RhoA embryo spreading and invasion [ 92 ] .\nUterine muscular contractions are required to expel the shed tissue lining during menses and the developed fetus during natural parturition [ 93 ] . Improper regulation of uterine muscle contractions can lead to preterm labor or retrograde menstruation, which is a common symptom of PCOS (see section “Roles of RhoA signaling in polycystic ovarian syndrome (PCOS)”). RhoA is the primary regulator of myosin light chain kinase phosphorylation, which directly regulates smooth muscle contraction  via  ROCK-mediated inhibition of myosin light chain phosphatase [ 93 ] . Oxytocin is a neuropeptide produced in the hypothalamus and stored in the posterior pituitary gland. During labor, the fetus exerts pressure on the cervix to trigger the release of oxytocin, the primary molecule released during labor [ 94 ] . The released oxytocin travels through the blood to the myometrium to activate the oxytocin receptor OXTR. OXTR is upregulated in the uterine fundus and upper segment and slightly upregulated in the lower uterine segment approaching parturition. RhoA is a key downstream signaling molecule that mediates oxytocin-induced myosin light chain phosphorylation and subsequent uterine contractions during labor [ 94 , 95 ] . The RhoA inhibitor simvastatin inhibited oxytocin-induced uterine contractions in a dose-dependent manner in non-pregnant and pregnant rats, as well as during parturition and on postpartum day 1 [ 96 ] , and oxytocin-induced contractility of rat myometrial strips was negated by the ROCK inhibitor Y-27632 [ 97 ] . These studies demonstrated the essential role of RhoA/ROCK in mediating oxytocin-induced uterine contractions. In contrast, lower expression of  RhoA  and  ROCK  during the earlier stages of pregnancy may contribute to uterine quiescence for maintaining pregnancy [ 96 ] .\nAlthough oxytocin is the primary endogenous molecule responsible for uterine contractility during parturition, the roles of other molecules have also been investigated. S1P and endothelin-1 (ET-1) both induce the contraction of rat myometrial strips and upregulate RhoA in the membrane fractions (active RhoA) [ 98 ] , which is consistent with the effect of ET-1 in promoting RhoA translocation from the cytoplasm to the membrane for ROCK activation in fibroblasts [ 99 ] . However, the ROCK inhibitor Y-27632 abolished S1P-induced myometrial contraction, while reducing approximately 50% of ET-1-induced myometrial contractions [ 98 ] , indicating that RhoA/ROCK signaling is essential for the contractile effect of S1P, while other mechanisms besides RhoA/ROCK are also involved in the contractile effect of ET-1. In addition, in ovariectomized rats, E2 treatment upregulates  RhoA  and  ROCK  mRNA levels and restores RhoA/ROCK-associated uterine contractile mechanisms [ 100 , 101 ] .\nInterestingly, the effect of the ROCK inhibitor H-1152 on uterine contractility was lower in d18 and d19 pregnant rat uterine tissue than in both non-pregnant and d21/d22 tissue, suggesting suppressed uterine RhoA/ROCK activity during pregnancy before the initiation of parturition [ 46 ] . While uterine  RhoA and ROCK  mRNA expression levels were comparable between non-pregnant and pregnant rats, ranging from gestation d16 to d22 to before labor,  Rnd-1  expression increased in late gestation and before labor [ 46 ] . RND proteins, including RND1, are unique Rho family G proteins that lack intrinsic GTPase activity and are therefore constitutively active. They activate p190 RhoGAP to antagonize RhoA activity [ 102 ] . Increased  Rnd-1  expression may suppress uterine RhoA activity and prevent preterm labor.\nThe functions of RhoA/ROCK in regulating uterine contractility have led to investigations of targeting this pathway for treating medical conditions with dysregulated uterine contractility, such as preterm labor and hypercholesterolemia-associated contractile deficits. Isoquinoline derivatives that are more potent ROCK inhibitors than the commonly used Y-27632 have been identified [ 103 ]  and can present a potential therapeutic approach for managing preterm labor complications. Hypercholesterolemia is commonly associated with uterine contractile deficits during pregnancy [ 104 ] . In gestation day 19 hypercholesterolemic mice, OXTR and RhoA protein levels were significantly reduced, and their uterine strips in the organ bath showed attenuated contractile responses to oxytocin [ 104 ] . These observations indicate the inhibitory effects of high cholesterol levels on RhoA activity through the downregulation of RhoA expression and its upstream oxytocin receptor. Therefore, activating the OXTR/RhoA signaling pathway could be a strategy to treat hypercholesterolemia-associated dampened uterine contractility.\nAdenomyosis is defined as an abnormal but confined growth of endometrial glands and stroma in the myometrium, which can alter uterine muscle contractions and cause dysmenorrhea or menorrhagia. The mRNA levels of  RhoA  and the downstream kinases  ROCK1  and  ROCK2  were increased, but those of  ARHGAP26 , a RhoGAP (GTPase activating protein, RhoA inactivator), were decreased in ectopic and eutopic human adenomyosis tissues compared to that in the normal endometrium [ 105 ] . However, the potential role of RhoA/ROCK signaling in adenomyosis has not yet been investigated.\nIntrauterine adhesions (Asherman’s syndrome) are a condition when scar tissues develop inside the uterus. RhoA and ROCK1 were upregulated, whereas ARHGAP29, a RhoGAP (RhoA inactivator) and a putative target mRNA of miR-1291, was downregulated in human endometrial tissues from patients with intrauterine adhesions compared with normal endometrium [ 106 ] . In the uterus of an intrauterine adhesion mouse model, the expression patterns of RhoA, ROCK1, and ARHGAP29 were similar to those in human intrauterine adhesion tissues; in addition, miR-1291 was upregulated, and treatment with an mi-R1291 antagomir upregulated ARHGAP29 expression and downregulated RhoA and ROCK1 expression, resulting in amelioration of endometrial fibrosis [ 106 ] . These observations implicate RhoA/ROCK1 in intrauterine adhesion and broader mechanisms, including epigenetic microRNAs, in the regulation of RhoA/ROCK signaling.\nEndometriosis is defined as the growth of uterine endometrial tissue outside the uterus. It affects approximately 10% of women worldwide and is a leading cause of infertility [ 107 ] . Compared with the eutopic endometrium, RhoA and associated downstream kinases ROCK1 and ROCK2 are upregulated in the ectopic endometrium (endometrium outside the uterus); they are also upregulated or show a trend of upregulation in the eutopic endometrium of patients with endometriosis compared with their expression in the endometrium of women without endometriosis. In cultured human eutopic endometrial epithelial cells (eutopic EECs), RhoA overexpression or knockdown led to enhanced or suppressed cell mobility, respectively; and in an endometriosis mouse model, abnormal activation of the RhoA/ROCK pathway was shown to mediate the effect of estrogen/estrogen receptor alpha (ERα)/ERK signaling in promoting the development of endometriosis [ 108 ] . The clinical implications of RhoA signaling in endometriosis remain to be investigated.\nUterine leiomyoma, or uterine fibroids, are characterized by noncancerous tumor growths that originate within the myometrium and are often coupled with aberrant estrogen signaling, representing a common source of clinical issues in women [ 109 ] . A defining characteristic of leiomyoma cells is the extensive network of disorganized ECM deposition [ 110 ] , and RhoA plays a role in ECM remodeling. An investigation of immortalized patient-matched myometrial and leiomyoma cells revealed that treatment with serum containing LPA resulted in a significant increase in active Rho protein levels, measured  via  the G-Lisa assay, in leiomyoma cells compared with those in myometrial control, with both serum starvation and anti-integrin β1 antibody treatment reducing active Rho levels [ 111 ] . The same group also found that immortalized leiomyoma cells plated on a rigid substrate had elevated levels of active RhoA compared to both myometrial control as well as leiomyoma and myometrial cells plated on a flexible substrate, indicating the attenuation of mechanical signaling in leiomyoma cells mediated by RhoA [ 112 ] .\nThe inhibition of RhoA pathway proteins may provide insights into leiomyoma development and potential treatment strategies. In immortalized leiomyoma cells induced to overexpress progesterone receptor isoform B (PRB) cultured on a stiff matrix, PRB activation was found to be mediated through RhoA activity  via  a luciferase assay, and treatment with the RhoGEF inhibitor A13 and ROCK inhibitor Y-27632 significantly decreased PRB activity (no notable PRB activity was detected on flexible matrices in both control and leiomyoma groups) [ 113 ] . Increased PRB activity may modulate estrogen signaling, while estrogen signaling has the potential to upregulate PR expression, which could have impacts in the reported mechanical attenuation. Using immortalized primary leiomyoma cells, it was revealed that simvastatin inhibits RhoA activation, resulting in a reduction in collagen gel contraction, which relies heavily on the developed ECM and cell–matrix interactions [ 114 ] . Simvastatin also results in degradation of the ECM in human leiomyoma xenografts in mice [ 115 ] . Treatment with fasudil, a potent Rho-kinase inhibitor and vasodilator, resulted in slight inhibition of collagen gel contraction in 3D leiomyoma cultures, further supporting the involvement of Rho/ROCK signaling in leiomyoma [ 116 ] . Together, these studies illustrate the interconnectedness of RhoA/ROCK signaling, the ECM, and uterine leiomyoma. Targeting RhoA/ROCK signaling could provide an important therapeutic strategy for women with uterine leiomyoma.\n\nThe cervix is the lower and narrower portion of the uterus connecting the corpus uterus to the vagina and undergoes dynamic changes throughout pregnancy, parturition, and the postpartum period. The cervix plays an especially vital role in childbirth because cervical dilation is crucial in vaginal deliveries. Premature or delayed cervical ripening can lead to early or post-term delivery, respectively. Analyses of RhoA and ROCK in the rat cervix during pregnancy, delivery, and postpartum revealed that  RhoA  mRNA levels were consistent throughout late pregnancy and delivery; however, RhoA protein levels were significantly decreased on pregnancy D20 and at parturition compared with those in non-pregnant cervix [ 117 ] . No change in  ROCK1  mRNA was found until pregnancy D20, following which there was a significant increase by D22 before parturition compared with the non-pregnant cervix, whereas  ROCK2  mRNA levels were consistent until parturition, where they significantly decreased compared with both previously investigated days as well as non-pregnant cervix. Both RhoA and ROCK1/2 protein levels showed a significant decrease at the time of parturition compared with both previously investigated days and non-pregnant cervix (except for RhoA protein, which was only different compared with non-pregnant cervix), which correlates to the time point with the least cervical resistance and may contribute to cervical ripening and delivery. RhoA and ROCK protein levels in the cervix return to baseline levels immediately after birth, suggesting their involvement in restoring cervical strength [ 117 ] . In the mouse cervix, both mRNA and protein levels of RhoA drastically decreased with advancing gestation compared with non-pregnant mouse cervix (<5% of control levels at D17 of pregnancy), suggesting transcriptional regulation of  RhoA  and potential species differences in RhoA regulation in the cervix, whereas RhoB was shown to have an opposite regulation trend compared with that of RhoA [ 118 ] . The upregulation of  RhoA  mRNA in the uterus and downregulation in the cervix of rats during parturition indicates the spatiotemporal regulation of RhoA in the female reproductive tract to confer its expected roles in increasing uterine contractions and decreasing cervical resistance for parturition.\nRhoA/ROCK-dependent NF-κB synthesis was shown to participate in lipopolysaccharide (LPS)-stimulated production of interleukin-8 (IL-8), which plays a role in cervical ripening [ 119 ] , and blocking the RhoA/ROCK pathway by C3 transferase exoenzyme or Y-27632 suppressed LPS-stimulated IL-8 production in cultured primary human cervical stromal cells [ 119 ] . The RhoA/ROCK pathway could be a potential therapeutic target to regulate cervical ripening and aid in the management of pre- or post-term deliveries.\nThe RhoA/ROCK pathway is also involved in the development and progression of cervical cancer. Western blot assays of cervical cancer specimens showed RhoA overexpression, and an investigation of RhoA overexpression in HeLa cell lines demonstrated increased proliferation and migration [ 120 ] . Dysregulation of RhoA regulators affect HeLa cell survival and invasion. Overexpression of BRACA1 interacting protein (BRIP1), which plays a role in DNA damage repair, promotes apoptosis and suppresses RhoA activation in HeLa cells, which can be reversed by LPA, a RhoA activator [ 121 ] . Overexpression of Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) activates RhoA, ROCK1, and pERM (phosphoezrin/radizin/moesin); promotes cell proliferation, cell migration, and epithelial-to-mesenchymal transition; and decreases cell apoptosis in cultured HeLa cells [ 122 ] . G12, a transforming oncogene, is upregulated in both HeLa and CaSki cervical carcinoma cell lines, and promotes cancer cell invasion, a process mediated by the RhoA/ROCK/JNK pathway [ 123 ] . RNA-binding protein with serine-rich domain 1 (RNPS1) is a regulator of the splicing process and is highly expressed in the cervical cancer cell lines HeLa, SiHa, and HDF. RNPS1 knockdown downregulated RhoA through alternative splicing [ 124 ] , suggesting that high levels of RNPS1 expression may contribute to elevated RhoA expression in cervical cancer cells. Parallel scenarios were also observed for the eukaryotic initiation factor elF5A2, which was upregulated in cervical cancer, and elF5A2 knockdown downregulated the expression of RhoA, ROCK1, and ROCK2, inhibited tumor growth and cell motility, and induced cell cycle arrest in the G1 phase [ 125 ] . Zinc finger X-linked duplicated family member (ZXDC) acts as an upstream activator of the RhoA/ROCK pathway by enabling the transcription of insulin-like growth factor IGF2BP3, which stabilizes  RhoA  mRNA for translation thereby increasing RhoA protein levels [ 126 ] . Based on GEO data from HeLa/SiHa cell lines and human cervical cancer tissues, ZXDC is significantly overexpressed in cervical cancer tissues compared with healthy controls, and cancer tissues with positive lymph node metastasis had significantly higher ZXDC expression than those without, indicating a potential role of ZXDC in the upregulation of RhoA in cervical cancer tissues [ 126 ] . Vascular endothelial growth factor C (VEGF-C) was also upregulated in cervical cancer tissues with metastasis, and its activation of the RhoA/ROCK2/Moesin pathway may contribute to actin remodeling and cell migration in cervical cancer metastasis [ 127 ] .\nSeveral studies have explored the therapeutic potential of RhoA/ROCK pathway regulators in cervical cancer. Inhibition of 6-phosphogluconate dehydrogenase (6PGD) using the inhibitor physcion (a natural anthraquinone derivative) was linked to the inhibition of tumor development, which involved the suppression of RhoA/Rac1 [ 128 ] . Sevoflurane, an anesthetic that enhances chemosensitivity and may have anticancer properties, inhibits RhoA/RAS downstream activities in HeLa, SiHa, and C-33A cells, resulting in decreased cell proliferation and migration [ 129 ] . The tumor suppressor miRNA-200b was shown to inhibit RhoA activity in a dual-luciferase reporter assay in HeLa cells, leading to reduced cell proliferation and enhanced apoptosis [ 130 ] . These studies support the use of RhoA inhibition as a strategy for treating cervical cancer.\n\nThe placenta is an essential organ that facilitates maternal blood supply to the fetus, regulating nutrition, gas exchange, and excretion of waste products and promoting fetal immunity. Placentation occurs when trophoblasts invade the maternal decidualized endometrium and migrate to the spiral arteries. ARHGEF18/p114RhoGEF is a tight junction–associated RhoA activator. It forms complexes with myosin II and regulates actomyosin contractility. Mice deficient in p114RhoGEF have abnormal placental development characterized by disrupted syncytiotrophoblast differentiation and fusion to form the fetomaternal barrier [ 131 ] . The involvement of RhoA was not specifically investigated in this study model, but PKA/CREB signaling and actomyosin remodeling were studied, which are coordinated by p114RhoGEF to promote trophoblast cell–cell fusion during placental morphogenesis [ 131 ] . Additional studies have implicated the association of RhoA in the placenta with pathological conditions.\nPreeclampsia is a serious medical condition mainly caused by inadequate spiral artery remodeling with the key symptoms being high blood pressure and proteinuria. Abnormally elevated RhoA expression appears to be a contributing factor in preeclampsia. RhoA mRNA and protein levels are upregulated in the third-trimester human placental tissues of patients with preeclampsia [ 132 ] , which suggests transcriptional upregulation of RhoA. Another study suggested that the accumulation of RhoA, a substrate of cullin 3-ring ubiquitin ligase, was owing to the decreased abundance of key components of cullin 3-ring ubiquitin ligase in the placentas and spiral arteries of patients with preeclampsia; notably, RhoA participates in enhanced vasoconstriction, which correlates with hypertension in patients with preeclampsia [ 133 ] . Interestingly, another study showed that the activity of Rac1, another member of the Rho family of GTPases, is significantly lower in the placentas of women with preeclampsia [ 134 ] . An  in vitro  study in human umbilical vein endothelial cell (HUVEC) lines indicated that overexpression of urothelial cancer-associated 1 (UCA1), a long non-coding RNA elevated in preeclampsia, led to increased activation of the RhoA/ROCK pathway [ 135 ] . Reduced S1P levels and sphingosine kinase 1 expression and activity were detected in the placenta of patients with preeclampsia. Moreover, in pregnant mice, inhibition of sphingosine kinase 1 activity during placentation suppresses placental S1P production and defective placentation, resulting in a preeclampsia phenotype [ 136 ] . This study also demonstrated the involvement of the S1P/S1P receptor 2 (S1PR2, a GPCR)/RhoA/ROCK signaling pathway in S1P-induced cell invasion of HTR8/SVneo immortalized human trophoblast cells [ 136 ] . These studies suggest that abnormally elevated RhoA/ROCK signaling is a mechanism underlying the development of preeclampsia. Therefore, RhoA/ROCK is a potential therapeutic target. Fasudil, a potent Rho-kinase inhibitor and vasodilator, can attenuate soluble fms-like tyrosine kinase-1 (sFlt-1)-induced hypertension in pregnant mice through the RhoA/ROCK pathway and protect vascular endothelial cells from hypoxia/reoxygenation-induced apoptosis in HUVEC [ 137 ] . Although the etiology of preeclampsia could be multifaceted and difficult to specify, the symptoms of preeclampsia, such as hypertension, could potentially be alleviated using RhoA/ROCK inhibitors.\nPAS disorders are a group of serious placenta-related medical conditions characterized by excessive invasion of chorionic villi in the myometrium, leading to failure in the detachment of the placenta after delivery accompanied by severe hemorrhage during and after delivery. In extravillous trophoblastic cells of PAS placentae, CXCL12 and its receptor CXCR4/CXCR7 are upregulated and their expression levels are closely related to the invasion depth of trophoblastic cells; in cultured HTR8/SVneo immortalized human trophoblast cells, the expression levels of each of these three genes  via  gene silencing or overexpression correlated with cell invasion as well as the expression levels of RhoA, Rac1, and Cdc42; furthermore, Rock inhibitor Y‐27632 inhibited cell invasion induced by overexpression of CXCL12, CXCR4, or CXCR7 [ 138 ] . The role of Rho/ROCK signaling in the excessive invasion of chorionic villi in the myometrium remains to be investigated.\n\nThe vagina connects the cervix to the exterior through an opening. It is primarily composed of smooth muscles and a mucus membrane, which encompasses an acidic vaginal canal. Muscle relaxation is closely associated with positive sexual responses. An ovariectomized rat model demonstrated that RhoA/ROCK functionality and expression were decreased in the absence of estrogen, leading to vaginal smooth muscle relaxation and increased sexual response; however, in the presence of testosterone,  RhoA  gene expression remained stable, but docking of RhoA to the plasma membrane of distal vaginal cells was inhibited [ 101 ] . Therefore, the link between RhoA and endogenous hormones should be further explored to elucidate their impact on sexual arousal and function.\nSmooth muscle constriction in the internal pudendal artery decreases blood flow to the vagina. RhoA and one of its upstream regulators, ET-1, a vasoconstrictor peptide, were upregulated in the internal pudendal artery of diabetic Goto-Kakizaki female rats, which had increased constriction sensitivity and decreased sexual response; however, RhoA was not found to be the sole downstream target of ET-1 in blood flow regulation. However, treatment of the internal pudendal and clitoral arteries from rats with the ROCK inhibitor Y-27632 reduced the contractile effect caused by ET-1, demonstrating the necessity of Rho/ROCK-mediated contractions for regulating blood flow [ 139 , 140 ] . Further research should be conducted on comorbidities such as diabetes and their impact on RhoA in vaginal and clitoral blood flow.\nPOP is a phenomenon in which pelvic organs erroneously bulge through the vagina, characterized by aberrant smooth muscle bundles that are smaller and fewer in number in the vaginal wall [ 141 ] . While little work has been done examining the role of RhoA in POP, the predefined activities of RhoA in smooth muscle contractility indicate its potential regulatory role in the development of this pathology. Uterosacral ligaments are essential for supporting pelvic organs and are often a site of disrepair throughout aging and the postpartum period. Recombinant human collagen, a novel treatment for female pelvic floor dysfunction and POP, has been shown to act by upregulating the GTP-bound form of RhoA, as well as ROCK activation, working to remodel the ECM and repair uterosacral ligaments  via  increased cell adhesion, migration, and endogenous collagen synthesis [ 142 ] . RhoA, ROCK1/2, and S1P receptors 2 and 3 (upstream regulators of the RhoA/ROCK pathway) are upregulated in the vaginal tissues of human patients with POP [ 39 ] , which might be a compensatory response to maintain the elasticity and strength of the vagina under duress. Fine-tuning of vaginal smooth muscle relaxation and contraction  via  RhoA/ROCK modulation is necessary to ensure healthy vaginal function and proper pelvic floor maintenance.\n\nThe mammary glands are vital postpartum structures that depend heavily on developmental regulation during both puberty and pregnancy for successful function. Branching morphogenesis is initiated during puberty by growth hormones and estrogen, among other factors, and the actions of progesterone and prolactin during pregnancy initiate further development of mammary glands [ 143 ] .  In vitro  murine studies have shown that mammary branching morphogenesis is partly dependent on ROCK-mediated RhoA signaling, with the inhibition of RhoA/ROCK signaling resulting in a disorganized, hyperbranched phenotype and incomplete myoepithelial cell coverage [ 144 , 145 ] . P190A-RhoGAP and P190B-RhoGAP are negative regulators of RhoA. Terminal end buds (TEB) are vital structures that form at the end of a developing mammary duct and drive ductal development. Overexpression or deletion of these isoforms in mice disrupts TEB architecture and proliferation and alters mammary gland differentiation and E2 and P4 receptor expression [ 146 – 148 ] . Knockout of the RhoGEF Net1, an activator of RhoA, has also been shown to result in impaired murine mammary gland development, with reduced phosphorylation of myosin light chain and myosin light chain phosphatase, along with decreased ERα expression [ 149 ] . The Human Protein Atlas indicates that RhoA mRNA and protein expression in human mammary glands is primarily localized to adipocytes with modest glandular expression [ 76 ] . These studies demonstrate that the regulation of RhoA, such as the coordination of other structural developments, is essential for mammary gland development. Species-specific regulation of RhoA activity and functions in the mammary gland requires further investigation.\nMammary glands provide nutrition for newborn infants. In cultured murine HC11 mammary epithelial cells, RhoA is proposed as a parathyroid hormone–related protein (PTHrP) serotonylation target involved in newborn mineralization to release calcium during lactation [ 150 ] . High activation levels of the RhoA pathway also impair prolactin signaling in first-/second-passage primary murine mammary epithelial cells [ 151 ] . The proposed anti-lactogenic pathways of RhoA include RhoA/ROCK kinase inhibition of insulin signaling in primary cultured mid-pregnant murine mammary epithelial cells [ 152 ]  and Rho downstream effector protein kinase N1 (PKN1) signaling impairing tight junction sealing  via  glucocorticoid blockage, as well as limiting milk secretion after parturition in a mouse model with constitutive activation of PKN1 [ 153 ] . The overexpression and uncontrolled behavior of RhoA  in vitro  may provide insights into a vital therapeutic pathway to ensure successful nutrient delivery to infants and establish and maintain sufficient lactogenesis.\nBreast cancer is one of the most common types of cancer among women, and metastasis to other organs and tissue systems represents one of the most prominent causes of cancer-related mortality. Breast cancer subtypes can be primarily categorized into four subvariants: luminal A-type, luminal B-type, HER-2 overexpressing, and basal-like, with categorization based on varying expression patterns of ERα, PR, and HER-2 [ 154 ] . Both upregulation and downregulation of RhoA appear to have implications in breast cancer.\nRhoA (along with Rac1 and Cdc42) expression was considerably higher in malignant human breast tissues than in normal breast tissues from the same patients; however, the mRNA and protein levels of RhoA appeared to be uncorrelated, and delayed RhoA degradation in malignant breast tissues could be the reason [ 155 ] . A similar phenomenon of increased RhoA expression in malignancy was observed in 4T1 (derived from spontaneous murine mammary tumors) and MCF10 (immortalized human mammary epithelium) cell lines. In these cell lines, miR-182 expression was positively correlated with metastasis. Missing in metastasis (MIM) is a target of miR-182 and a suppressor of RhoA. The inhibition of MIM by miR-182 leads to increased actin stress fiber formation, a hallmark of RhoA activity, and an increased metastatic phenotype [ 156 ] . Confirmatory examination with the RhoA inhibitor C3 exoenzyme showed an effect similar to that of phenotypic MIM expression, blocking cell invasion and cytoskeletal remodeling [ 156 ] . Other microRNAs, such as miR-146a [ 157 ]  and miR-155 [ 158 ] , have also been reported to regulate RhoA expression/activity in breast cancer cell lines. In addition, the overexpression of RhoGDI, the protein responsible for the sequestration of inactive RhoA, results in a reversible increase in breast cancer resistance to etoposide and doxorubicin [ 158 ] . Indeed, RhoA activation promotes drug resistance in breast cancer cells [ 159 ] , and RhoA/ROCK signaling mediates the effects of doxorubicin on cell migration and invasion  via  increased myosin light chain 2 phosphorylation in MCF7 and BT-474 cells [ 160 ] . RhoA has also been shown to play a role in the cellular functions of disheveled 2 (Dvl2) and disheveled-associated activator of morphogenesis 1 (Daam1), which are downstream molecules of Wnt5a signaling [ 161 ] , and diaphanous-related formin-3 (DIAPH3), a cytoskeletal regulator and RhoA inhibitor, with decreased expression in luminal A-type, luminal B-type, HER-2 + , and triple-negative (no ERα, PR, or HER-2) clinical human breast cancer tissues [ 162 ] .\nAlthough activation of RhoA signaling is positively correlated with breast cancer formation and progression in most cases, RhoA knockdown in a syngeneic triple-negative breast cancer murine model had no impact on tumor formation or proliferation; however, this knockdown significantly increased the rate of invasion into lymph nodes and metastasis to the lungs [ 163 ] . The results of this study contrast with those observed in primary human tissues and both human and murine cell lines and may represent either an alternative pathway of RhoA activity in breast cancer or a species-specific difference revealed  in vivo . These reports reveal the complexity of developing therapeutic strategies for treating not only breast cancers but all types of cancers in different patients, because of individual variations, such as variations in oncogenic origin, genetics, and environment. Nevertheless, RhoA signaling has cellular effects on cancer cells, and targeting RhoA signaling (inhibiting or activating)  via  microRNAs or chemotherapy remains a potential strategy for cancer therapy.\n\nThis is the first comprehensive review of studies of RhoA in the FRS. It provides an emerging picture of the spatiotemporal functions of RhoA in both physiological and pathological conditions. Significant knowledge gaps still exist regarding the cell type–specific and stage-specific functions of RhoA in the FRS, especially  in vivo . Cell type–specific RhoA upstream and downstream signaling pathways associated with each RhoA function remain to be elucidated. Spatial transcriptomic technology enables the spatiotemporal transcriptome analysis of genes. However, this technology has limitations in studying RhoA, as RhoA activity is mainly regulated at the post-transcriptional level. The Cre-loxP system is a powerful tool for studying the loss of cell type–specific gene functions. However, the use of this technology for studying cell type–specific functions of molecules in RhoA signaling pathways in FRS has been limited owing to the ubiquitous expression of many molecules in RhoA signaling pathways, including RhoA, as well as by the lack of suitable cell-type–specific Cre mice. For example, during our attempts to generate a uterine epithelial–specific RhoA conditional knockout mouse model using two commonly used Cre mice,  Wnt7A Cre  mice and  Ltf Cre  mice, the former yielded  RhoA f/f Wnt7A Cre  mice with hydrocephalus and postnatal death [ 164 ] , while the latter was genetically impossible owing to the localization of both  RhoA  and  Ltf  on chromosome 9. As the FRS is under hormonal control, the pathways through which RhoA signaling mutually interacts with hormonal signaling is an important area that requires extensive investigation. RhoA has been implicated in many pathological conditions in the FRS; therefore, targeting RhoA signaling to develop therapeutic strategies needs to be explored.\n\nThe authors thank the Office of the Vice President for Research, Interdisciplinary Toxicology Program, Department of Physiology and Pharmacology at the University of Georgia and the National Institutes of Health (NIH) for their financial support.\n\nS.V., Y.L., and X.Y. conceived the idea; S.V. initiated the drafting of the manuscript as her senior thesis; J.K.S. wrote the manuscript with assistance from all coauthors; J.K.S. drew the figures; all authors contributed to the construction and revision of the manuscript; and X.Y. supervised the entire process.\n\nThis study was supported by the following grants: NIH R03HD097384, R03HD100652, and R01HD114750 to X.Y.\n\nAll authors declare no conflicts of interest.\n\nNot applicable.","source_license":"CC0","license_restricted":false}