Functional genomics analysis identifies loss of HNF1B function as a cause of Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a congenital condition characterized by aplasia or hypoplasia of the uterus and vagina in women with a 46,XX karyotype. This condition can occur as type I when isolated or as type II when associated with extragenital anomalies including kidney and skeletal abnormalities. The genetic basis of MRKH syndrome remains unexplained and several candidate genes have been proposed to play a role in its etiology, including HNF1B, LHX1 and WNT4. Here, we conducted a microarray analysis of 13 women affected by MRKH syndrome, resulting in the identification of chromosomal changes, including the deletion at 17q12, which contains both HNF1B and LHX1. We focused on HNF1B for further investigation due to its known association with, but unknown etiological role in, MRKH syndrome. We ablated Hnf1b specifically in the epithelium of the Müllerian ducts in mice and found that this caused hypoplastic development of the uterus, as well as kidney anomalies, closely mirroring the MRKH type II phenotype. Using single-cell RNA sequencing of uterine tissue in the Hnf1b-ablated embryos, we analyzed the molecules and pathways downstream of Hnf1b, revealing a dysregulation of processes associated with cell proliferation, migration and differentiation. Thus, we establish that loss of Hnf1b function leads to an MRKH phenotype and generate the first mouse model of MRKH syndrome type II. Our results support the investigation of HNF1B in clinical genetic settings of MRKH syndrome and shed new light on the molecular mechanisms underlying this poorly understood condition in women's reproductive health.

Extracellular vesicles as a potential delivery platform for CRISPR-Cas based therapy in epithelial ovarian cancer.

Ovarian Cancer (OC) is the most common gynecological malignancy and the eighth most diagnosed cancer in females worldwide. Presently, it ranks as the fifth leading cause of cancer-related mortality among patients globally. Major factors contributing to the lethality of OC worldwide include delayed diagnosis, chemotherapy resistance, high metastatic rates, and the heterogeneity of subtypes. Despite continuous efforts to develop novel targeted therapies and chemotherapeutic agents, challenges persist in the form of OC resistance and recurrence. In the last decade, CRISPR-Cas-based genome editing has emerged as a powerful tool for modifying genetic and epigenetic mechanisms, holding potential for treating numerous diseases. However, a significant challenge for therapeutic applications of CRISPR-Cas technology is the absence of an optimal vehicle for delivering CRISPR molecular machinery into targeted cells or tissues. Recently, extracellular vesicles (EVs) have gained traction as potential delivery vehicles for various therapeutic agents. These heterogeneous, membrane-derived vesicles are released by nearly all cells into extracellular spaces. They carry a molecular cargo of proteins and nucleic acids within their intraluminal space, encased by a cholesterol-rich phospholipid bilayer membrane. EVs actively engage in cell-to-cell communication by delivering cargo to both neighboring and distant cells. Their inherent ability to shield molecular cargo from degradation and cross biological barriers positions them ideally for delivering CRISPR-Cas ribonucleoproteins (RNP) to target cells. Furthermore, they exhibit higher biocompatibility, lower immunogenicity, and reduced toxicity compared to classical delivery platforms such as adeno-associated virus, lentiviruses, and synthetic nanoparticles. This review explores the potential of employing different CRISPR-Cas systems to target specific genes in OC, while also discussing various methods for engineering EVs to load CRISPR components and enhance their targeting capabilities.

Generation and mutational analysis of a transgenic mouse model of human SRY.

SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage the pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive testis determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRY's three domains (N-terminal, HMG box, and C-terminal) and performed a detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and present a versatile and powerful system in which to functionally analyze variations of SRY including known and novel pathogenic variants found in DSD.

The Embryological Landscape of Mayer-Rokitansky-Kuster-Hauser Syndrome: Genetics and Environmental Factors.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a disorder caused by Müllerian ducts dysgenesis affecting 1 in 5000 women with a typical 46,XX karyotype. The etiology of MRKH syndrome is complex and largely unexplained. Familial clustering suggests a genetic component and the spectrum of clinical presentations seems consistent with an inheritance pattern characterized by incomplete penetrance and variable expressivity. Mutations of several candidate genes have been proposed as possible causes based on genetic analyses of human patients and animal models. In addition, studies of monozygotic twins with discordant phenotypes suggest a role for epigenetic changes following potential exposure to environmental compounds. The spectrum of clinical presentations is consistent with intricate disruptions of shared developmental pathways or signals during early organogenesis. However, the lack of functional validation and translational studies have limited our understanding of the molecular mechanisms involved in this condition. The clinical management of affected women, including early diagnosis, genetic testing of MRKH syndrome, and the implementation of counseling strategies, is significantly impeded by these knowledge gaps. Here, we illustrate the embryonic development of tissues and organs affected by MRKH syndrome, highlighting key pathways that could be involved in its pathogenesis. In addition, we will explore the genetics of this condition, as well as the potential role of environmental factors, and discuss their implications to clinical practice.

Lhx8 ablation leads to massive autophagy of mouse oocytes associated with DNA damage.

Following proliferation of oogonia in mammals, great numbers of germ cells are discarded, primarily by apoptosis, while the remainder form primordial follicles (the ovarian reserve) that determine fertility and reproductive lifespan. More massive, rapid, and essentially total loss of oocytes, however, occurs when the transcription factor Lhx8 is ablated-though the cause and mechanism of germ cell loss from the Lhx8-/- ovaries has been unknown. We found that Lhx8-/- ovaries maintain the same number of germ cells throughout embryonic development; rapid decrease in the pool of oocytes starts shortly before birth. The loss results from activation of autophagy, which becomes overwhelming within the first postnatal week, with extracellular matrix proteins filling the space previously occupied by follicles to produce a fibrotic ovary. Associated with this process, as early as a few days before birth, Lhx8-/- oocytes failed to repair DNA damage-which normally occurs when meiosis is initiated during embryonic development; and DNA damage repair genes were downregulated throughout the oocyte short lifespan. Based on gene expression analyses and morphological changes, we propose a model in which lineage-restricted failure of DNA repair triggers germ cell autophagy, causing premature depletion of the ovarian reserve in Lhx8-/- mice.

WNT/ß-catenin and p27/FOXL2 differentially regulate supporting cell proliferation in the developing ovary.

Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising ß-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/ß-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/ß-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.

FOXL2 modulates cartilage, skeletal development and IGF1-dependent growth in mice.

BACKGROUND: Haploinsufficiency of the FOXL2 transcription factor in humans causes Blepharophimosis/Ptosis/Epicanthus Inversus syndrome (BPES), characterized by eyelid anomalies and premature ovarian failure. Mice lacking Foxl2 recapitulate human eyelid/forehead defects and undergo female gonadal dysgenesis. We report here that mice lacking Foxl2 also show defects in postnatal growth and embryonic bone and cartilage formation. METHODS: Foxl2 (-/-) male mice at different stages of development have been characterized and compared to wild type. Body length and weight were measured and growth curves were created. Skeletons were stained with alcian blue and/or alizarin red. Bone and cartilage formation was analyzed by Von Kossa staining and immunofluorescence using anti-FOXL2 and anti-SOX9 antibodies followed by confocal microscopy. Genes differentially expressed in skull vaults were evaluated by microarray analysis. Analysis of the GH/IGF1 pathway was done evaluating the expression of several hypothalamic-pituitary-bone axis markers by RT-qPCR. RESULTS: Compared to wild-type, Foxl2 null mice are smaller and show skeletal abnormalities and defects in cartilage and bone mineralization, with down-regulation of the GH/IGF1 axis. Consistent with these effects, we find FOXL2 expressed in embryos at 9.5 dpc in neural tube epithelium, in head mesenchyme near the neural tube, and within the first branchial arch; then, starting at 12.5 dpc, expressed in cartilaginous tissue; and at PO and P7, in hypothalamus. CONCLUSIONS: Our results support FOXL2 as a master transcription factor in a spectrum of developmental processes, including growth, cartilage and bone formation. Its action overlaps that of SOX9, though they are antagonistic in female vs male gonadal sex determination but conjoint in cartilage and skeletal development.

Dynamics of the ovarian reserve and impact of genetic and epidemiological factors on age of menopause.

The narrow standard age range of menopause, ∼50 yr, belies the complex balance of forces that govern the underlying formation and progressive loss of ovarian follicles (the "ovarian reserve" whose size determines the age of menopause). We show here the first quantitative graph of follicle numbers, distinguished from oocyte counts, across the reproductive lifespan, and review the current state of information about genetic and epidemiological risk factors in relation to possible preservation of reproductive capacity. In addition to structural X-chromosome changes, several genes involved in the process of follicle formation and/or maintenance are implicated in Mendelian inherited primary ovarian insufficiency (POI), with menopause before age 40. Furthermore, variants in a largely distinct cohort of reported genes-notably involved in pathways relevant to atresia, including DNA repair and cell death-have shown smaller but additive effects on the variation in timing of menopause in the normal range, early menopause (age <45), and POI. Epidemiological factors show effect sizes comparable to those of genetic factors, with smoking accounting for about 5% of the risk of early menopause, equivalent to the summed effect of the top 17 genetic variants. The identified genetic and epidemiological factors underline the importance of early detection of reproductive problems to enhance possible interventions.

FOXL2 transcriptionally represses Sf1 expression by antagonizing WT1 during ovarian development in mice.

Steroidogenic factor 1 (SF1; Ad4BP/NR5A1) plays key roles in gonadal development. Initially, the Sf1 gene is expressed in mouse fetal gonads of both sexes, but later is up-regulated in testes and down-regulated in ovaries. While Sf1 expression is activated and maintained by Wilms tumor 1 (WT1) and LIM homeobox 9 (LHX9), the mechanism of sex-specific regulation remains unclear. We hypothesized that Sf1 is repressed by the transcription factor Forkhead box L2 (FOXL2) during ovarian development. In an in vitro system (TM3 cells), up-regulation of Sf1 by the WT1 splice variant WT1-KTS was antagonized by FOXL2, as determined by quantitative RT-PCR. Using reporter assays, we localized the Sf1 proximal promoter region involved in this antagonism to a 674-bp interval. A conserved FOXL2 binding site was identified in this interval by in vitro chromatin immunoprecipitation. Introducing mutations into this site abolished negative regulation by FOXL2 in reporter assays. Finally, in Foxl2-null mice, Sf1 expression was increased 2-fold relative to wild-type XX fetal gonads. Our results support the hypothesis that FOXL2 negatively regulates Sf1 expression by antagonizing WT1-KTS during early ovarian development in mice.

Notch gain of function in mouse periocular mesenchyme downregulates FoxL2 and impairs eyelid levator muscle formation, leading to congenital blepharophimosis.

Notch signaling is pivotal for the morphogenesis and homeostasis of many tissues. We found that aberrant Notch activation in mouse neural-crest-derived periocular mesenchymal cells (POMCs), which contribute to the formation of corneal and eyelid stroma, results in blepharophimosis. Compound transgenic mice overexpressing the Notch1 intracellular domain (N1-ICD) in POMCs (POMC(N1-ICD)) showed relatively minor effects on the cornea, but increased cell apoptosis and decreased cell proliferation during eyelid morphogenesis. Eyelid closure at E15.5 and eyelid formation at birth were incomplete. In further analyses, overexpression of N1-ICD impaired eyelid levator smooth muscle formation by downregulating the transcription factor FoxL2. This is similar to the effect of haploinsufficiency of FOXL2 in humans, which results in type II BPES (blepharophimosis, ptosis and epicanthus inversus syndrome). In vitro studies showed that FoxL2 expression is augmented by a low dose of N1-ICD but was downregulated by a high dose, depending on the extent of Hes-1 and Hey-1 activation. Moreover, transfection of CMV-FoxL2 enhanced α-SMA promoter activity. These data strongly imply that a physiologically low level of Notch1 is crucial for proper FoxL2 expression in POMCs, which is, in turn, essential for Müeller muscle formation and normal eyelid development.

Ovarian absence: a systematic literature review and case series report.

Ovarian absence is an uncommon condition that most frequently presents unilaterally. Several etiologies for the condition have been proposed, including torsion, vascular accident, and embryological defect. A systematic review was conducted to describe the clinical presentation of ovarian absence, as well as its associations with other congenital anomalies, through a systematic search of Cochrane Library, ClinicalTrials.gov, Google Scholar, Ovid Embase, Ovid Medline, PubMed, Scopus, and Web of Science. Exclusion criteria included cases with suspicion for Differences of Sex Development, lack of surgically-confirmed ovarian absence, and karyotypes other than 46XX. Our search yielded 12,120 citations, of which 79 studies were included. 10 additional studies were found by citation chasing resulting in a total 113 cases including two unpublished cases presented in this review. Abdominal/pelvic pain (30%) and infertility/subfertility (19%) were the most frequent presentations. Ovarian abnormalities were not noted in 28% of cases with pre-operative ovarian imaging results. Approximately 17% of cases had concomitant uterine abnormalities, while 22% had renal abnormalities. Renal abnormalities were more likely in patients with uterine abnormalities (p < 0.005). Torsion or vascular etiology was the most frequently suspected etiology of ovarian absence (52%), followed by indeterminate (27%) and embryologic etiology (21%). Most cases of ovarian absence are likely attributable to torsion or vascular accidents, despite many references to the condition as "agenesis" in the literature. Imaging may fail to correctly diagnose ovarian absence, and diagnostic laparoscopy may be preferable in many cases as genitourinary anatomy and fertility considerations can be assessed during the procedure. Fertility is likely minimally or not affected in women with unilateral ovarian absence.

Antagonistic regulation of Cyp26b1 by transcription factors SOX9/SF1 and FOXL2 during gonadal development in mice.

Sex determination in fetal germ cells depends on a balance between exposure to retinoic acid (RA) and the degradation of RA achieved by the testis-specific expression of the catabolic cytochrome P450 enzyme, CYP26B1. Therefore, identification of factors regulating the expression of the Cyp26b1 gene is an important goal in reproductive biology. We used in situ hybridization to demonstrate that Cyp26b1 and transcription factor genes steroidogenic factor-1 (Sf1) and Sry-related HMG box 9 (Sox9) are coexpressed in Sertoli cells, whereas Cyp26b1 and Sf1 are coexpressed in Leydig cells in mouse fetal testes. In the mouse gonadal somatic cell line TM3, transfection of constructs expressing SOX9 and SF1 activated Cyp26b1 expression, independently of the positive regulator RA. In embryonic gonads deficient in SOX9 or SF1, Cyp26b1 expression was decreased relative to wild-type (WT) controls, as measured by quantitative RT-PCR (qRT-PCR). Furthermore, qRT-PCR showed that Cyp26b1 up-regulation by SOX9/SF1 was attenuated by the ovarian transcription factor Forkhead box L2 (FOXL2) in TM3 cells, whereas in Foxl2-null mice, Cyp26b1 expression in XX gonads was increased ∼20-fold relative to WT controls. These data support the hypothesis that SOX9 and SF1 ensure the male fate of germ cells by up-regulating Cyp26b1 and that FOXL2 acts to antagonize Cyp26b1 expression in ovaries.

Functional Analysis of Mmd2 and Related PAQR Genes During Sex Determination in Mice.

INTRODUCTION: Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. Mmd2 is expressed during the sex-determining period in XY but not XX gonads, suggesting a specific role in testis development. METHODS: We used CRISPR to generate mouse strains deficient in Mmd2 and its 2 closely related PAQR family members, Mmd and Paqr8, which are also expressed during testis development. Following characterization of Mmd2 expression in the developing testis, we studied sex determination in embryos from single knockout as well as Mmd2;Mmd and Mmd2;Paqr8 double knockout lines using quantitative RT-PCR and immunofluorescence. RESULTS: Analysis of knockout mice deficient in Sox9 and Nr5a1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, fetal testis development progressed normally in Mmd2-null embryos. To determine if other genes might have compensated for the loss of Mmd2, we analyzed Paqr8 and Mmd-null embryos and confirmed that in both knockout lines, sex determination occurred normally. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. DISCUSSION: These results may reflect functional redundancy among PAQR factors, or their dispensability in gonadal development. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

Foxl2 functions in sex determination and histogenesis throughout mouse ovary development.

BACKGROUND: Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women. In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit. RESULTS: Following Foxl2 loss, early testis genes (including Inhbb, Dhh, and Sox9) and several novel ovarian genes were consistently dysregulated during embryonic development. In the absence of Foxl2, expression changes affecting a large fraction of pathways were opposite those observed in Wnt4-null ovaries, reinforcing the notion that these genes have complementary actions in ovary development. Loss of one copy of Foxl2 revealed strong gene dosage sensitivity, with molecular anomalies that were milder but resembled ovaries lacking both Foxl2 alleles. Furthermore, a Foxl2 transgene disrupted embryonic testis differentiation and increased the levels of key female markers. CONCLUSION: The results, including a comprehensive principal component analysis, 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for roles in sex determination independent of FOXL2 (e.g., the transcription factors IRX3 and ZBTB7C) and in the generation of the ovarian reserve downstream of FOXL2 (e.g., the cadherin-domain protein CLSTN2 and the sphingomyelin synthase SGMS2). The gene inventory is a first step toward the identification of the full range of pathways with partly autonomous roles in ovary development, and thus provides a framework to analyze the genetic bases of female fertility.

Identification of Candidate Genes for Mayer-Rokitansky-Küster-Hauser Syndrome Using Genomic Approaches.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a disorder of sex development which affects 1 in 4,500 females and is characterized by agenesis of müllerian structures, including the uterus, cervix, and upper vagina. It can occur in isolation (type 1) or in conjunction with various anomalies (type 2), with a subset of these comprising müllerian, renal, and cervicothoracic abnormalities (MURCS) association. The genetic causes of MRKH have been investigated previously yielding limited results, with massive parallel sequencing becoming increasingly utilized. We sought to identify genetic contributions to MRKH using a combination of microarray and whole exome sequencing (WES) on a cohort of 8 unrelated women with MRKH and MURCS. WES data were analysed using a candidate gene approach to identify potential contributing variants. Microarray analysis identified a 0.6-Mb deletion in the previously implicated 16p11.2 region in a patient with MRKH type 2. WES revealed 16 rare nonsynonymous variants in MRKH candidate genes across the cohort. These included variants in several genes, such as LRP10 and DOCK4, associated with disorders with müllerian anomalies. Further functional studies of these variants will help to delineate their biological significance and expand the genotypic spectrum of MRKH.

Author Correction: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9.

Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Variants in two key genes, SRY and its target SOX9, are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the regulatory elements underlying this have not been identified in humans. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata.

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml(-1)) and by HeLa, HT29 and JM cells with IC50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml(-1), all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.

Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae).

From the caudices of the Passifloraceae Adenia lanceolata and A. stenodactyla, two lectins called lanceolin and stenodactylin, respectively, were purified by affinity chromatography on CL Sepharose 6B. The lectins are glycoproteins with M(r) 61,243 (lanceolin) and 63,131 (stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties, with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from A. volkensii. The lectins agglutinate red blood cells, inhibit protein synthesis both by a cell-free system and by whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A). They are highly toxic to cells, in which they induce apoptosis, and to mice, with LD(50)s 8.16 microg/kg (lanceolin) and 2.76 microg/kg (stenodactylin) at 48 h. Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosome-inactivating proteins and are amongst the most potent toxins of plant origin.