Patterns of admixture and introgression in a mosaic Mytilus galloprovincialis and Mytilus edulis hybrid zone in SW England.

The study of hybrid zones offers important insights into speciation. Earlier studies on hybrid populations of the marine mussel species Mytilus edulis and Mytilus galloprovincialis in SW England provided evidence of admixture but were constrained by the limited number of molecular markers available. We use 57 ancestry-informative SNPs, most of which have been mapped genetically, to provide evidence of distinctive differences between admixed populations in SW England and asymmetrical introgression from M. edulis to M. galloprovincialis. We combine the genetic study with analysis of phenotypic traits of potential ecological and adaptive significance. We demonstrate that hybrid individuals have brown mantle edges unlike the white or purple in the parental species, suggesting allelic or non-allelic genomic interactions. We report differences in gonad development stage between the species consistent with a prezygotic barrier between the species. By incorporating results from publications dating back to 1980, we confirm the long-term stability of the hybrid zone despite higher viability of M. galloprovincialis. This stability coincides with a dramatic change in temperature of UK coastal waters and suggests that these hybrid populations might be resisting the effects of global warming. However, a single SNP locus associated with the Notch transmembrane signalling protein shows a markedly different pattern of variation to the others and might be associated with adaptation of M. galloprovincialis to colder northern temperatures.

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism.

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2 It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.

A simple strategy for sample annotation error detection in cytometry datasets.

Mislabeling samples or data with the wrong participant information can affect study integrity and lead investigators to draw inaccurate conclusions. Quality control to prevent these types of errors is commonly embedded into the analysis of genomic datasets, but a similar identification strategy is not standard for cytometric data. Here, we present a method for detecting sample identification errors in cytometric data using expression of human leukocyte antigen (HLA) class I alleles. We measured HLA-A*02 and HLA-B*07 expression in three longitudinal samples from 41 participants using a 33-marker CyTOF panel designed to identify major immune cell types. 3/123 samples (2.4%) showed HLA allele expression that did not match their longitudinal pairs. Furthermore, these same three samples' cytometric signature did not match qPCR HLA class I allele data, suggesting that they were accurately identified as mismatches. We conclude that this technique is useful for detecting sample-labeling errors in cytometric analyses of longitudinal data. This technique could also be used in conjunction with another method, like GWAS or PCR, to detect errors in cross-sectional data. We suggest widespread adoption of this or similar techniques will improve the quality of clinical studies that utilize cytometry.

Effects of growth rate, cell size, motion, and elemental stoichiometry on nutrient transport kinetics.

Nutrient acquisition is a critical determinant for the competitive advantage for auto- and osmohetero- trophs alike. Nutrient limited growth is commonly described on a whole cell basis through reference to a maximum growth rate (Gmax) and a half-saturation constant (KG). This empirical application of a Michaelis-Menten like description ignores the multiple underlying feedbacks between physiology contributing to growth, cell size, elemental stoichiometry and cell motion. Here we explore these relationships with reference to the kinetics of the nutrient transporter protein, the transporter rate density at the cell surface (TRD; potential transport rate per unit plasma-membrane area), and diffusion gradients. While the half saturation value for the limiting nutrient increases rapidly with cell size, significant mitigation is afforded by cell motion (swimming or sedimentation), and by decreasing the cellular carbon density. There is thus potential for high vacuolation and high sedimentation rates in diatoms to significantly decrease KG and increase species competitive advantage. Our results also suggest that Gmax for larger non-diatom protists may be constrained by rates of nutrient transport. For a given carbon density, cell size and TRD, the value of Gmax/KG remains constant. This implies that species or strains with a lower Gmax might coincidentally have a competitive advantage under nutrient limited conditions as they also express lower values of KG. The ability of cells to modulate the TRD according to their nutritional status, and hence change the instantaneous maximum transport rate, has a very marked effect upon transport and growth kinetics. Analyses and dynamic models that do not consider such modulation will inevitably fail to properly reflect competitive advantage in nutrient acquisition. This has important implications for the accurate representation and predictive capabilities of model applications, in particular in a changing environment.

Proteomic analysis of eggs from Mytilus edulis females differing in mitochondrial DNA transmission mode.

Many bivalves have an unusual mechanism of mitochondrial DNA (mtDNA) inheritance called doubly uniparental inheritance (DUI) in which distinctly different genomes are inherited through the female (F genome) and male (M genome) lineages. In fertilized eggs that will develop into male embryos, the sperm mitochondria remain in an aggregation, which is believed to be delivered to the primordial germ cells and passed to the next generation through the sperm. In fertilized eggs that will develop into female embryos, the sperm mitochondria are dispersed throughout the developing embryo and make little if any contribution to the next generation. The frequency of embryos with the aggregated or dispersed mitochondrial type varies among females. Previous models of DUI have predicted that maternal nuclear factors cause molecular differences among unfertilized eggs from females producing embryos with predominantly dispersed or aggregated mitochondria. We test this hypothesis using females of each of the two types from a natural population. We have found small, yet detectable, differences of the predicted type at the proteome level. We also provide evidence that eggs of females giving the dispersed pattern have consistently lower expression for different proteasome subunits than eggs of females giving the aggregated pattern. These results, combined with those of an earlier study in which we used hatchery lines of Mytilus, and with a transcriptomic study in a clam that has the DUI system of mtDNA transmission, reinforce the hypothesis that the ubiquitin-proteasome system plays a key role in the mechanism of DUI and sex determination in bivalves. We also report that eggs of females giving the dispersed pattern have higher expression for arginine kinase and enolase, enzymes involved in energy production, whereas ferritin, which is involved in iron homeostasis, has lower expression. We discuss these results in the context of genetic models for DUI and suggest experimental methods for further understanding the role of these proteins in DUI.

Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines.

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4(+)/IFN-γ(+) T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4(+)/IFN-γ(+)-inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4(+) T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

Multiple hypothesis testing in proteomics: a strategy for experimental work.

In quantitative proteomics work, the differences in expression of many separate proteins are routinely examined to test for significant differences between treatments. This leads to the multiple hypothesis testing problem: when many separate tests are performed many will be significant by chance and be false positive results. Statistical methods such as the false discovery rate method that deal with this problem have been disseminated for more than one decade. However a survey of proteomics journals shows that such tests are not widely implemented in one commonly used technique, quantitative proteomics using two-dimensional electrophoresis. We outline a selection of multiple hypothesis testing methods, including some that are well known and some lesser known, and present a simple strategy for their use by the experimental scientist in quantitative proteomics work generally. The strategy focuses on the desirability of simultaneous use of several different methods, the choice and emphasis dependent on research priorities and the results in hand. This approach is demonstrated using case scenarios with experimental and simulated model data.

Identification and characterization of highly expressed proteins in sperm cells of the marine mussel Mytilus edulis.

Proteomic analysis on sperm has been restricted to only a few model organisms. We present here a 2DE PAGE proteome map of sperm cells from a nonmodel organism, the marine mussel Mytilus edulis, a free-spawning marine invertebrate with external fertilization. Ninety-six protein spots showing high expression were selected and of these 77 were successfully identified by nESI-MS analysis. Many of the identifications are relevant to sperm cell physiology and mtDNA functioning. The results and proteomics approach used are discussed in relation to their potential for advancing understanding of the unusual system of mtDNA inheritance described in Mytilus spp., and for the testing of evolutionary hypotheses pertaining to the role of fertilization in the speciation process.

Multiple hypothesis testing in proteomics: A strategy for experimental work.

In quantitative proteomics work, the differences in expression of many separate proteins are routinely examined to test for significant differences between treatments. This leads to the multiple hypothesis testing problem: when many separate tests are performed many will be significant by chance and be false positive results. Statistical methods such as the false discovery rate (FDR) method that deal with this problem have been disseminated for more than one decade. However a survey of proteomics journals shows that such tests are not widely implemented in one commonly used technique, quantitative proteomics using two-dimensional electrophoresis (2-DE). We outline a selection of multiple hypothesis testing methods, including some that are well known and some lesser known, and present a simple strategy for their use by the experimental scientist in quantitative proteomics work generally. The strategy focuses on the desirability of simultaneous use of several different methods, the choice and emphasis dependent on research priorities and the results in hand. This approach is demonstrated using case scenarios with experimental and simulated model data.

Proteomic analysis of F1 hybrids and intermediate variants in a Littorina saxatilis hybrid zone.

Proteomic analysis was carried out on the Crab (upper-shore) and Wave (lower-shore) ecotypes of Littorina saxatilis from a hybrid zone at Silleiro Cape, Spain. Proteome profiles of individual snails were obtained. Protein expression in F1 hybrid snails bred in the laboratory and snails with intermediate shell phenotypes collected from the mid-shore were compared with Crab and Wave ecotypes using analytical approaches used to study dominance. Multivariate analysis over many protein spots showed that the F1 snails are distinct from both ecotypes but closer to the Wave ecotype. The intermediate snails are highly variable, some closer to the Crab and others to the Wave ecotype. Considered on a protein by protein basis, some proteins are significantly closer in expression to the Crab and others to the Wave ecotype for both F1 and intermediate snails. Furthermore, a significant majority of proteins were closer in expression to the Wave ecotype for the F1, consistent with the multivariate analysis. No such significant majority toward either the Crab or Wave ecotype was observed for the intermediate snails. The closer similarity of F1 and Wave ecotype expression patterns could be the result of similar selective pressures in the similar mid-shore and low-shore environments. For a significantly larger number of proteins, intermediate snails were closer in expression to the ecotype having the lower expression, for both Crab and Wave ecotypes. This is somewhat unexpected as lower expression might be expected to be an indication of impairment of function and lower fitness. Proteomic analysis could be important for the identification of candidate proteins useful for gaining improved understanding of adaptation and barriers to gene flow in hybrid zones.

Flow cytometry: an alternative method for direct quantification of antigens adsorbed to aluminum hydroxide adjuvant.

Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 µg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods.

Genetic variation underlying protein expression in eggs of the marine mussel Mytilus edulis.

Study of the genetic basis of gene expression variation is central to attempts to understand the causes of evolutionary change. Although there are many transcriptomics studies estimating genetic variance and heritability in model organisms such as humans there is a lack of equivalent proteomics studies. In the present study, the heritability underlying egg protein expression was estimated in the marine mussel Mytilus. We believe this to be the first such measurement of genetic variation for gene expression in eggs of any organism. The study of eggs is important in evolutionary theory and life history analysis because maternal effects might have profound effects on the rate of evolution of offspring traits. Evidence is presented that the egg proteome varies significantly between individual females and that heritability of protein expression in mussel eggs is moderate to high suggesting abundant genetic variation on which natural selection might act. The study of the mussel egg proteome is also important because of the unusual system of mitochondrial DNA inheritance in mussels whereby different mitochondrial genomes are transmitted independently through female and male lineages (doubly uniparental inheritance). It is likely that the mechanism underlying this system involves the interaction of specific egg factors with sperm mitochondria following fertilization, and its elucidation might be advanced by study of the proteome in females having different progeny sex ratios. Putative identifications are presented here for egg proteins using MS/MS in Mytilus lines differing in sex ratio. Ontology terms relating to stress response and protein folding occur more frequently for proteins showing large expression differences between the lines. The distribution of ontology terms in mussel eggs was compared with those for previous mussel proteomics studies (using other tissues) and with mammal eggs. Significant differences were observed between mussel eggs and mussel tissues but not between the two types of eggs.

The COVID-19 immune landscape is dynamically and reversibly correlated with disease severity.

BACKGROUNDDespite a rapidly growing body of literature on coronavirus disease 2019 (COVID-19), our understanding of the immune correlates of disease severity, course, and outcome remains poor.METHODSUsing mass cytometry, we assessed the immune landscape in longitudinal whole-blood specimens from 59 patients presenting with acute COVID-19 and classified based on maximal disease severity. Hospitalized patients negative for SARS-CoV-2 were used as controls.RESULTSWe found that the immune landscape in COVID-19 formed 3 dominant clusters, which correlated with disease severity. Longitudinal analysis identified a pattern of productive innate and adaptive immune responses in individuals who had a moderate disease course, whereas those with severe disease had features suggestive of a protracted and dysregulated immune response. Further, we identified coordinate immune alterations accompanying clinical improvement and decline that were also seen in patients who received IL-6 pathway blockade.CONCLUSIONThe hospitalized COVID-19 negative cohort allowed us to identify immune alterations that were shared between severe COVID-19 and other critically ill patients. Collectively, our findings indicate that selection of immune interventions should be based in part on disease presentation and early disease trajectory due to the profound differences in the immune response in those with mild to moderate disease and those with the most severe disease.FUNDINGBenaroya Family Foundation, the Leonard and Norma Klorfine Foundation, Glenn and Mary Lynn Mounger, and the National Institutes of Health.

Exploring evolution of maximum growth rates in plankton.

Evolution has direct and indirect consequences on species-species interactions and the environment. However, Earth systems models describing planktonic activity invariably fail to explicitly consider organism evolution. Here we simulate the evolution of the single most important physiological characteristic of any organism as described in models-its maximum growth rate (µm). Using a low-computational-cost approach, we incorporate the evolution of µm for each of the plankton components in a simple Nutrient-Phytoplankton-Zooplankton -style model such that the fitness advantages and disadvantages in possessing a high µm evolve to become balanced. The model allows an exploration of parameter ranges leading to stresses, which drive the evolution of µm. In applications of the method we show that simulations of climate change give very different projections when the evolution of µm is considered. Thus, production may decline as evolution reshapes growth and trophic dynamics. Additionally, predictions of extinction of species may be overstated in simulations lacking evolution as the ability to evolve under changing environmental conditions supports evolutionary rescue. The model explains why organisms evolved for mature ecosystems (e.g. temperate summer, reliant on local nutrient recycling or mixotrophy), express lower maximum growth rates than do organisms evolved for immature ecosystems (e.g. temperate spring, high resource availability).

Introducing the Endotype Concept to Address the Challenge of Disease Heterogeneity in Type 1 Diabetes.

The clinical diagnosis of new-onset type 1 diabetes has, for many years, been considered relatively straightforward. Recently, however, there is increasing awareness that within this single clinical phenotype exists considerable heterogeneity: disease onset spans the complete age range; genetic susceptibility is complex; rates of progression differ markedly, as does insulin secretory capacity; and complication rates, glycemic control, and therapeutic intervention efficacy vary widely. Mechanistic and immunopathological studies typically show considerable patchiness across subjects, undermining conclusions regarding disease pathways. Without better understanding, type 1 diabetes heterogeneity represents a major barrier both to deciphering pathogenesis and to the translational effort of designing, conducting, and interpreting clinical trials of disease-modifying agents. This realization comes during a period of unprecedented change in clinical medicine, with increasing emphasis on greater individualization and precision. For complex disorders such as type 1 diabetes, the option of maintaining the "single disease" approach appears untenable, as does the notion of individualizing each single patient's care, obliging us to conceptualize type 1 diabetes less in terms of phenotypes (observable characteristics) and more in terms of disease endotypes (underlying biological mechanisms). Here, we provide our view on an approach to dissect heterogeneity in type 1 diabetes. Using lessons from other diseases and the data gathered to date, we aim to delineate a roadmap through which the field can incorporate the endotype concept into laboratory and clinical practice. We predict that such an effort will accelerate the implementation of precision medicine and has the potential for impact on our approach to translational research, trial design, and clinical management.

CT043, a protective antigen that induces a CD4+ Th1 response during Chlamydia trachomatis infection in mice and humans.

Despite several decades of intensive studies, no vaccines against Chlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4(+) Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4(+) Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 from C. trachomatis human isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate against C. trachomatis infection.

The consequences of sample pooling in proteomics: an empirical study.

Pooling of samples in proteomics experiments might help overcome resource constraints when many individuals are analysed. The measured biological variation should be reduced giving increased power to detect treatment differences. Pooling has been advocated in microarray work but there are few tests of its potential in proteomics. In this study, we examine three issues on which the success of the pooling approach might hinge and provide evidence that: (i) the protein expression in a pool matches the mean expression of the individuals making up the pool for the majority of proteins, although for some proteins the pool expression is different; (ii) the biological variance between pools is reduced compared with that between individuals, as predicted in theory, but this reduction is not as large as expected. A practical consequence of this is that power could be reduced; (iii) proteins detectable in individual samples are usually but not always visible when samples are pooled. We conclude that pooling of samples in proteomics work is a valid and potentially valuable procedure but consideration should be given to these issues in experimental design.

Non-linear dose-response of DNA-reactive genotoxins: recommendations for data analysis.

Until recently, there has only been a limited amount of data available on the kinetics of mutation induction in the low dose region of exposure. In our publication Doak et al. [S.H. Doak, G.J. Jenkins, G.E. Johnson, E. Quick, E.M. Parry, J.M. Parry, Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens, Cancer Res. 67 (2007) 3904-3911] we showed that the two alkylating agents methyl-methanesulfonate (MMS) and ethyl-methanesulfonate (EMS) possess non-linear dose-response curves with no observed effect levels (NOEL) for mutation or chromosomal damage in vitro. These experiments were carried out in the AHH-1 human lymphoblastoid cell line, using the hypoxanthine phosphoribosyl transferase (HPRT) assay and the cytokinesis-block micronucleus (CBMN) assay, respectively. We have now carried out more advanced statistical analyses to define threshold values, which is critical as it has a dramatic impact on hazard and risk assessment. To do this, we re-analysed the data to see if the linear model or a more complex model (hockey stick or quadratic) gave a significant better fit of the data. For both EMS and MMS cytokinesis-block micronucleus data sets, the hockey stick model gave the most significant fit. The same was true for EMS, MMS and surprisingly ethylnitrosourea (ENU) in the HPRT assay in human AHH-1 cells. However, methylnitrosourea (MNU) was linear in both assays. These further analyses have shown that EMS and MMS have clear thresholds for both gene mutation and chromosome damage, as does ENU for gene mutation in AHH-1 cells. MNU was linear for gene and chromosome mutation and so was ENU for chromosome mutations at the concentrations tested. These findings correlate closely with those in vivo findings of Gocke et al. [E. Gocke, L. Müller, In vivo studies in the mouse to define a threshold for the genotoxicity of EMS and ENU, Mutat. Res. (this issue)] and together these data show a true threshold for EMS both in vitro and in vivo. In this report, we will discuss the approaches that were taken to investigate potential threshold dose-response curves for DNA-reactive genotoxic compounds, with recommendations for further studies.

RNA-seq coupled to proteomic analysis reveals high sperm proteome variation between two closely related marine mussel species.

Speciation mechanisms in marine organisms have attracted great interest because of the apparent lack of substantial barriers to genetic exchange in marine ecosystems. Marine mussels of the Mytilus edulis species complex provide a good model to study mechanisms underlying species formation. They hybridise extensively at many localities and both pre- and postzygotic isolating mechanisms may be operating. Mussels have external fertilisation and sperm cells should show specific adaptations for survival and successful fertilisation. Sperm thus represent key targets in investigations of the molecular mechanisms underlying reproductive isolation. We undertook a deep transcriptome sequencing (RNA-seq) of mature male gonads and a 2DE/MS-based proteome analysis of sperm from Mytilus edulis and M. galloprovincialis raised in a common environment. We provide evidence of extensive expression differences between the two mussel species, and general agreement between the transcriptomic and proteomic results in the direction of expression differences between species. Differential expression is marked for mitochondrial genes and for those involved in spermatogenesis, sperm motility, sperm-egg interactions, the acrosome reaction, sperm capacitation, ATP reserves and ROS production. Proteins and their corresponding genes might thus be good targets in further genomic analysis of reproductive barriers between these closely related species. SIGNIFICANCE: Model systems for the study of fertilization include marine invertebrates with external fertilisation, such as abalones, sea urchins and mussels, because of the ease with which large quantities of gametes released into seawater can be collected after induced spawning. Unlike abalones and sea urchins, hybridisation has been reported between mussels of different Mytilus spp., which thus makes them very appealing for the study of reproductive isolation at both pre- and postzygotic levels. There is a lack of empirical proteomic studies on sperm samples comparing different Mytilus species, which could help to advance this study. A comparative analysis of sperm proteomes across different taxa may provide important insights into the fundamental molecular processes and mechanisms involved in reproductive isolation. It might also contribute to a better understanding of sperm function and of the adaptive evolution of sperm proteins in different taxa. There is now growing evidence from genomics studies that multiple protein complexes and many individual proteins might have important functions in sperm biology and the fertilisation process. From an applied perspective, the identification of sperm-specific proteins could also contribute to the improved understanding of fertility problems and as targets for fertility control.