Is endometriosis due to evolutionary maladaptation?

Similar to diabetes and unlike many pathogen-induced diseases, endometriosis is likely a result of maladaptation to the evolutionary heritage of humans. The objective of this article is to review the literature and improve understanding of the evolutionary factors behind endometriosis, leading to more effective prevention and treatment approaches. In primates, spontaneous decidualization of the endometrium evolved to ensure optimal implantation of a limited number of early embryos, unlike many non-primates which depend on early embryos to induce decidualization and subsequent pregnancy. Spontaneous decidualization results in menstrual bleeding when embryo implantation does not occur, and endometriosis is commonly believed to be caused by retrograde menstruation. Although direct evidence is lacking, it is likely that hunter-gatherer women experienced fewer menstrual periods due to pregnancy shortly after menarche, followed by repeated pregnancies and lactation. However, the mismatch between the evolved uterine physiology and rapid societal changes has led to modern women delaying pregnancy and experiencing numerous menstrual periods, potentially increasing the incidence of endometriosis. The symptoms of endometriosis are often managed by suppressing menstruation through systemic hormonal treatments, but these may have side effects. For patients with a family history of endometriosis or in the early stages of the disease, intrauterine devices releasing progesterone locally could prevent uterine bleeding and the development of endometriosis while preserving fertility and minimizing side effects.

Gonacin: A germ cell-derived hormone with glucogenic, orexigenic, and gonadal activities.

Fish require abundant nutrients to generate a large number of eggs for spawning. Based on the evolutionary conservation of human FBN2 and its C-terminal placensin-like sequences in fish, we identified a peptide hormone gonacin (GONAdal Cell placensIN) and found its high expression in early-stage germ cells in the ovary and testis of zebrafish. We demonstrated that gonacin is essential for food intake, glucose release, and ovarian development in zebrafish. Similar expression patterns and functions of gonacin were also demonstrated in rainbow trout. Gonacin represents the first hormone secreted by germ cells with endocrine functions in vertebrates, bridging the energy homeostasis and reproduction.

Out of step societal and Darwinian adaptation during evolution is the cause of multiple women's health issues.

During human evolution, major changes in our societal conditions and environment took place without sufficient time for concomitant genetic alterations, leading to out of step adaptation and diseases in women. We first discuss recent societal adaptation mismatch (menstrual bleeding; increases in cancers of reproductive organs, endometriosis; mother's nursing; polycystic ovarian syndrome; transgenerational epigenetic modifications), followed by Darwinian out of step adaptation (labor difficulties; sex chromosomes, human diseases and sex disparity in genomic DNA). We discuss the evolutionary basis of menstrual bleeding, followed by recent increases in cancers of reproductive organs and endometriosis. The importance of breastfeeding by mothers is also emphasized. Earlier onset of menarche, decreased rates of childbirths and breastfeeding resulted in increased number of menstrual cycles in a lifetime, coupled with excess estrogen exposure and incessant ovulation, conditions that increased the susceptibility to mammary and uterine cancers as well as ovarian epithelial cancer and endometriosis. Shorter lactation duration in mothers also contributed to more menstrual cycles. We further discuss the evolutionary basis of the prevalent polycystic ovary syndrome. During the long-term Darwinian evolution, difficulties in childbirth evolved due to a narrowed pelvis, our upright walking and enlarged fetal brain sizes. Because there are 1.5% genomic DNA differences between woman and man, it is of significance to investigate sex-specific human physiology and diseases. In conclusion, understanding out of step adaptation during evolution could allow the prevention and better management of female reproductive dysfunction and diseases.

Spatial topological analysis of sympathetic neurovascular characteristic of acupoints in Ren meridian using advanced tissue-clearing and near infrared II imaging.

Acupuncture has been used for treating various medical conditions in traditional Chinese medicine. Both manual and electro-acupuncture stimulate specific acupoints to obtain local and systemic biological effects, but the underlying mechanisms remain unclear. Here, we used three-dimensional tissue-clearing technology to study acupoints on the Ren meridian of mice to reveal the distribution, density, branching, and relationships between blood vessels and nerves. Using topological Mapper methods, we found that sympathetic neurovascular networks were denser in the CV 4 acupoint compared with surrounding non-acupoints. Furthermore, high resolution in vivo real-time vascular imaging using the near infrared-II probe LZ-1105 demonstrated increased blood flow in the CV 4 acupoint compared with neighboring non-acupoints after manual or electro-acupuncture. Consistent with earlier findings, our research indicated that acupuncture could enhance local blood flow, and our high-resolution 3D images show for the first time the important role of sympathetic neurovascular networks in the CV 4 acupoint.

Hippo signaling disruption and ovarian follicle activation in infertile patients.

The Hippo signaling pathway, which is important in organ size regulation, is present in organisms from the fly to mammals. Disruption of the Hippo signaling pathway leads to increased nuclear translocation of the effector Yes-associated protein (YAP), resulting in the expression of cystein-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) growth factors and baculoviral inhibitors of apoptosis repeat containing (BIRC) apoptosis inhibitors to increase organ sizes. Furthermore, genome-wide knockdown of genes in insect cells demonstrated that actin polymerization promoted nuclear translocation of YAP. In the mammalian ovary, we demonstrated the expression of Hippo signaling pathway genes and showed that ovarian fragmentation increased actin polymerization, leading to YAP nuclear translocation and increased expression of cystein-rich 61, CCN growth factors and BIRC apoptosis inhibitors, followed by enhanced follicle growth. Here we summarize evidence suggesting the role of mechanical stress on follicle growth in the ovary and describe recent use of ovary-damaging procedures to treat ovarian infertility. Ovarian fragmentation, together with in vitro incubation with Akt-stimulating drugs, formed the basis of an in vitro activation (IVA) therapy to treat patients with premature ovarian insufficiency, whereas ovarian fragmentation alone (drug-free IVA) was successful in treating patients with premature ovarian insufficiency with recent menses cessation. For middle-aged women with poor ovarian responses and diminished ovarian reserve, drug-free IVA was also effective in promoting follicle growth for infertility treatment. In addition, an in vivo follicle activation approach based on laparoscopic ovarian incision showed promise for patients with resistant ovary syndrome. With initial success using mechanical disruption approaches, future investigation could evaluate possibilities to refine mechanical methods and to locally administer actin polymerization-enhancing drugs for ovarian infertility treatment.

Discovery of polypeptide ligand-receptor pairs based on their co-evolution.

Sequencing diverse genomes allowed the tracing of orthologous and paralogous genes to understand the co-evolution of polypeptide ligands and receptors. This review documents the discovery of several polypeptide ligands and their cognate receptors mainly expressed in the reproductive tissue using evolutionary genomics. We discussed the sub-functionization of paralogs and co-evolution of ligand-receptor families. Based on the conserved signaling among paralogous receptors and common knock-out phenotypes of ligand-receptor pairs, relationships between relaxin family peptides and leucine-rich repeat-containing, G protein-coupled receptors (LGR) were revealed. We also described the identification of a novel paralogous glycoprotein hormone thyrostimulin and design of a long-acting FSH. Human stresscopin and stresscopin-related peptide, paralogous to CRH, were also identified based on the conserved signaling pathways. Recently, a novel ligand placensin expressed in human placenta was found based on the paralogous relationship with a metabolic hormone asprosin. Placensin likely contributes to stage-dependent increases in insulin resistance during human pregnancy and its elevated secretion was associated with gestational diabetes mellitus. Although many ligands were predicted based on sequence signatures, ligands of shorter sequences have not been identified, together with many "orphan" receptors without known ligands. Future development of tools for predicting ligands and high throughput assays to identify ligand-receptor pairs based on ligand binding and/or signal transduction could advance hormone-based physiology and pathophysiology.

Drug-free in-vitro activation of follicles for infertility treatment in poor ovarian response patients with decreased ovarian reserve.

RESEARCH QUESTION: The recently developed in-vitro activation (IVA) approach provides a promising infertility treatment for patients with premature ovarian insufficiency. The IVA method promotes growth of residual ovarian follicles following ovarian tissue fragmentation leading to Hippo signalling disruption, together with in-vitro incubation with Akt stimulators. As poor ovarian response (POR) patients with decreased ovarian reserve (DOR) have multiple secondary follicles, this study tested whether Hippo signalling disruption alone using in-vitro ovarian cortical fragmentation, followed by autologous grafting, was sufficient to promote follicle growth. DESIGN: A case series study. RESULTS: In 9 out of 11 POR patients with DOR treated with a simplified IVA procedure, increases in antral follicle numbers in multiple growth waves were detected following FSH treatment. Subsequent injection with human chorionic gonadotrophin allowed retrieval of more mature oocytes for IVF (median antral follicle counts before and after IVA per ovarian stimulation: 1.0 versus 2.6) with 68.7% fertilization rates and 56.9% showing high-quality embryonic development. One natural conception and 16 embryo transfers in five patients resulted in one live birth, two ongoing pregnancies and one miscarriage. Three additional patients and the miscarriage patient have cryopreserved embryos for future transfer. CONCLUSIONS: The present drug-free IVA approach may be suitable for POR patients with DOR, as it increased the number of antral follicles. The procedure also eliminated the need for 2-day incubation with drugs and required only one surgery. This approach could allow the retrieval of more oocytes in middle-aged women to achieve higher pregnancy rates and deserves proper evaluation in future randomized controlled trials.

Imaging the ovary.

During each reproductive cycle, the ovary exhibits tissue remodelling and cyclic vasculature changes associated with hormonally regulated folliculogenesis, follicle rupture, luteal formation and regression. However, the relationships among different types of follicles and corpora lutea are unclear, and the role of ovarian vasculature in folliculogenesis and luteal dynamics has not been extensively investigated. Understanding of ovarian physiology and pathophysiology relies upon elucidation of ovarian morphology and architecture. This paper summarizes the literature on traditional approaches to the imaging of ovarian structures and discusses recent advances in ovarian imaging. Traditional in-vivo ultrasound, together with histological and electron microscopic approaches provide detailed views of the ovary at organ, tissue and molecular levels. However, in-vivo imaging is limited to antral and larger follicles whereas histological imaging is mainly two-dimensional in nature. Also discussed are emerging approaches in the use of near-infrared fluorophores to image follicles in live animals to detect preantral follicles as well as visualizing ovarian structures using CLARITY in fixed whole ovaries to elucidate three-dimensional interrelationships among follicles, corpora lutea and ovarian vasculature. Advances in ovarian imaging techniques provide new understanding of ovarian physiology and allow for the development of better tools to diagnose ovarian pathophysiology.

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P <0.01) between the length of the follicular capillaries and volume of the follicular wall.

CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.

Optimal distribution of heterogeneous organelles and cell types within an organ is essential for physiological processes. Unique for the ovary, hormonally regulated folliculogenesis, ovulation, luteal formation/regression and associated vasculature changes lead to tissue remodeling during each reproductive cycle. Using the CLARITY approach and marker immunostaining, we identified individual follicles and corpora lutea in intact ovaries. Monitoring lifetime changes in follicle populations showed age-dependent decreases in total follicles and percentages of advanced follicles. Follicle development from primordial to preovulatory stage was characterized by 3 × 105-fold increases in volume, decreases in roundness, and decreased clustering of same stage follicles. Construction of follicle-vasculature relationship maps indicated age- and gonadotropin-dependent increases in vasculature and branching surrounding follicles. Heterozygous mutant mice with deletion of hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter showed defective ovarian vasculature and decreased ovulatory responses. Unilateral intrabursal injection of axitinib, an inhibitor of VEGF receptors, retarded neo-angiogenesis that was associated with defective ovulation in treated ovaries. Our approach uncovers unique features of ovarian architecture and essential roles of vasculature in organizing follicles to allow future studies on normal and diseased human ovaries. Similar approaches could also reveal roles of neo-angiogenesis during embryonic development and tumorigenesis.

Activation of dormant follicles: a new treatment for premature ovarian failure?

PURPOSE OF REVIEW: Premature ovarian failure (POF) is diagnosed by amenorrhea before 40 years of age. Owing to exhaustion of follicles in POF ovaries, egg donation is the only option. Although menstrual cycles cease in POF patients, some of them still contain residual dormant follicles in ovaries. Recently, we developed a new infertility treatment and named it as in-vitro activation (IVA), which enables POF patients to conceive using their own eggs by activation of residual dormant follicles. Here, we summarize data showing the potential of IVA as a new infertility treatment for POF patients. RECENT FINDINGS: Transgenic mouse studies revealed that the stimulation of phosphatidylinositol-3-kinase-AKT-forkhead box O3 pathway activated dormant primordial follicles. In murine and human ovaries, the phosphatase and tensin homolog inhibitors and phosphatidylinositol-3-kinase activators were demonstrated to activate dormant primordial follicles in in-vitro cultures. Subsequent studies showed that ovarian fragmentation suppressed Hippo signaling pathway, leading to ovarian follicle growth. Combining these two methods in an IVA approach followed by ovarian tissue autotransplantation, successful follicle growth, and pregnancies were reported in POF patients. Currently, two healthy babies were delivered, together with two additional pregnancies. SUMMARY: IVA treatment is a potential infertility therapy for POF patients who have residual follicles.

Apela Regulates Fluid Homeostasis by Binding to the APJ Receptor to Activate Gi Signaling.

Apela (APJ early endogenous ligand, also known as elabela or toddler) is a recently discovered peptide hormone. Based on genetic studies in zebrafish, apela was found to be important for endoderm differentiation and heart development during embryogenesis. Although common phenotypes of apela and APJ-null zebrafish during embryonic development suggested that apela interacts with the APJ receptor, kinetics of apela binding to APJ and intracellular signaling pathways for apela remain unknown. The role of apela in adults is also uncertain. Using a chimeric apela ligand, we showed direct binding of apela to APJ with high affinity (Kd = 0.51 nm) and the ability of apelin, the known peptide ligand for APJ, to compete for apela binding. Apela, similar to apelin, acts through the inhibitory G protein pathway by inhibiting forskolin-stimulated cAMP production and by inducing ERK1/2 phosphorylation. In adult rats, apela is expressed exclusively in the kidney, unlike the wide tissue distribution of apelin. In vivo studies demonstrated the ability of apela to regulate fluid homeostasis by increasing diuresis and water intake. Dose-response studies further indicated that apela induces 2- and 5-fold higher maximal responses than apelin in ERK1/2 phosphorylation and diuresis/water intake, respectively. After designing an apela antagonist, we further demonstrated the role of endogenous ligand(s) in regulating APJ-mediated fluid homeostasis. Our results identified apela as a potent peptide hormone capable of regulating fluid homeostasis in adult kidney through coupling to the APJ-mediated Gi signaling pathway.

Intraovarian control of early folliculogenesis.

Although hormonal regulation of ovarian follicle development has been extensively investigated, most studies concentrate on the development of early antral follicles to the preovulatory stage, leading to the successful use of exogenous FSH for infertility treatment. Accumulating data indicate that preantral follicles are under stringent regulation by FSH and local intraovarian factors, thus providing the possibility to develop new therapeutic approaches. Granulosa cell-derived C-type natriuretic factor not only suppresses the final maturation of oocytes to undergo germinal vesicle breakdown before ovulation but also promotes preantral and antral follicle growth. In addition, several oocyte- and granulosa cell-derived factors stimulate preantral follicle growth by acting through wingless, receptor tyrosine kinase, receptor serine kinase, and other signaling pathways. In contrast, the ovarian Hippo signaling pathway constrains follicle growth and disruption of Hippo signaling promotes the secretion of downstream CCN growth factors capable of promoting follicle growth. Although the exact hormonal factors involved in primordial follicle activation has yet to be elucidated, the protein kinase B (AKT) and mammalian target of rapamycin signaling pathways are important for the activation of dormant primordial follicles. Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, promote the growth of preantral follicles in patients with primary ovarian insufficiency, leading to a new infertility intervention for such patients. Elucidation of intraovarian mechanisms underlying early folliculogenesis may allow the development of novel therapeutic strategies for patients diagnosed with primary ovarian insufficiency, polycystic ovary syndrome, and poor ovarian response to FSH stimulation, as well as for infertile women of advanced reproductive age.

Fertility: the role of mTOR signaling and KIT ligand.

Activation of a limited pool of diminishing ovarian follicles determines women's reproductive lifespan. A recent rodent study describes the role of mTOR signaling and KIT ligand in granulosa cells of primordial follicles for follicle activation and for reproductive lifespan regulation.

A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals.

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.

Correction: Actin Cytoskeleton Regulates Hippo Signaling.

[This corrects the article on p. e73763 in vol. 8.].

Actin cytoskeleton regulates Hippo signaling.

Hippo pathway controls the organ size by modulating cell proliferation and apoptosis. However, the upstream regulation of hippo signaling by actin cytoskeleton is not clear. To elucidate the role of actin as an upstream regulator of Hippo signaling, the levels of F (filamentous)-actin in cells were elevated using jasplakinolide, an actin-stabilizing drug. Induction of F-actin formation in HeLa cells resulted in decreased phosphorylation of YAP, a key effector molecule for Hippo signaling. The activated YAP is localized to the cell nucleus and YAP increase was associated with increased expression of downstream CCN growth factors CCN1/CYR61 and CCN2/CTGF. The effect of the actin-stabilizing drug was blocked when YAP levels were suppressed in YAP "knock-down" cells. In summary, using an actin-stabilizing drug we show that actin cytoskeleton is one of the upstream regulators of Hippo signaling capable of activating YAP and increasing its downstream CCN growth factors.

Evolution of a potential hormone antagonist following gene splicing during primate evolution.

Alternative splicing of genes generates novel mRNAs, leading to the evolution of new functional proteins. Cholecystokinin (CCK) induces the release of pancreatic enzymes and the contraction of the gallbladder to promote the digestion of fat and proteins. CCK activates two G-protein-coupled receptors, CCKA and CCKB. Here, we showed that a CCKsv (splicing variant), originated de novo during Catarrhini evolution by including a portion of intronic sequence of the CCK gene, encodes novel C-terminal peptide sequence followed by a new poly-adenylation signal. CCKsv is expressed in many human tissues and likely a secreted peptide retaining the original signal peptide and the N-terminal proteolytic processing signal, together with novel C-terminal sequences. Although CCKsv cannot activate CCK receptors, it partially inhibits the CRE- or SRF-driven reporter activities stimulated by wide type CCK-8 mediated by both CCK receptors. Co-treatment with CCKsv also partially antagonizes Ewing tumor cell growth stimulated by CCK-8. Our study provides an example of new peptide hormone antagonist evolution in primates.

Multi-functional norrin is a ligand for the LGR4 receptor.

Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways.

Oocyte-derived R-spondin2 promotes ovarian follicle development.

R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.