The urgent need for innovation in contraception.

Despite advancements in medicine over the past decades, there exists a significant unmet global need for new and improved contraceptive methods for men and women. The development of innovative contraceptives will be facilitated via advancements in biomedical science, biomedical engineering, and drug development technologies. This article describes the need for new methods, opportunities afforded by advancements in biomedical science, strategies being employed to advance innovative novel methods, value of drug development accelerators and the need for industry involvement to provide men and women worldwide greater reproductive autonomy.

The new contraceptive revolution: developing innovative products outside of industry†,‡.

A significant global unmet need for new contraceptive options for both women and men remains due to side effect profiles, medical concerns, and inconvenience of many currently available products. The pharmaceutical industry has largely abandoned early research and development for contraception and will not likely engage to bring new products to the market unless they have been significantly de-risked by showing promise in early phase clinical trials. This lack of interest by big pharma comes at a time when scientific and technological advances in biology and medicine are creating more opportunities than ever for the development of new and innovative drug products. Novel partnerships between the academic sector, small biotechnology companies, foundations, non-government organizations (NGOs), and the federal government could accelerate the development of new contraceptive products. We discuss the challenges and opportunities that we have encountered as an NGO with a mission to develop novel contraceptive products for low- and middle-income countries and how it differs from developing products for higher-income markets. We hope that our experiences and "lessons learned" will be of value to others as they proceed down the product development path, be it for female or male or for hormonal or nonhormonal contraceptives.

Discovery of 6-({4-[2-(4-tert-butylphenyl)-1H-benzimidazol-4-yl]piperazin-1-yl}methyl)quinoxaline (WAY-207024): an orally active antagonist of the gonadotropin releasing hormone receptor (GnRH-R).

A potent, highly insoluble, GnRH antagonist with a 2-phenyl-4-piperazinylbenzimidazole template and a quinoxaline-2,3-dione pharmacophore was modified to maintain GnRH antagonist activity and improve in vitro pharmaceutical properties. Structural changes to the quinoxaline-2,3-dione portion of the molecule resulted in several structures with improved properties and culminated in the discovery of 6-([4-[2-(4-tert-butylphenyl)-1H-benzimidazol-4-yl]piperazin-1-yl] methyl)quinoxaline (WAY-207024). The compound was shown to have excellent pharmacokinetic parameters and lowered rat plasma LH levels after oral administration.

Approaches to the identification of new nonhormonal targets for male contraception.

Fertility control is a global health issue with major personal and societal impact. Although, currently, there are several different options for contraception, the technologies behind these are antiquated, and the options for male-based contraception (i.e., withdrawal, condom and vasectomy) are inadequate. The genomic, proteomic and bioinformatic revolutions have provided new tools and new targets for contraceptive development, and the results of such approaches have identified gene products that play critical roles in male reproduction, thus expanding the array of potential targets for novel and innovative male-based contraceptives. This article will review the types of targets being considered in the development of nonhormonal male contraceptives and the technologies used to identify and validate these targets.

Stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and Sertoli cells.

Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes, and spermatids suggests the existence of precise programs of gene expression in these cells and in their neighboring somatic Sertoli cells. The objective of this study was to identify the genes that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, and XIII-XIV of the cycle were isolated by microdissection, whereas Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes, and round spermatids were purified from enzymatically dispersed testes. Microarray analysis by using Rat Genome 230 2.0 arrays identified 16,971 probe sets that recognized testicular transcripts, and 398 of these were identified as testis-specific. Expression of 1,286 probe sets were found to differ at least 4-fold between two cell types and also across the stages of the cycle. Pathway and annotated cluster analyses of those probe sets predicted that entire biological pathways and processes are regulated cyclically in specific cells. Important among these are the cell cycle, DNA repair, and embryonic neuron development. Taken together, these data indicate that stage-regulated gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells.

2-phenyl-4-piperazinylbenzimidazoles: orally active inhibitors of the gonadotropin releasing hormone (GnRH) receptor.

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has shown positive clinical results in numerous reproductive tissue disorders such as endometriosis, prostate cancer and others. Traditional therapy has been limited to peptide agonists and antagonists. Recently, small molecule GnRH antagonists have emerged as potentially new treatments. This article describes the discovery of 2-phenyl-4-piperazinylbenzimidazoles as small molecule GnRH antagonists with nanomolar potency in in vitro binding and functional assays, excellent bioavailability (rat %F>70) and demonstrated oral activity in a rat model having shown significant serum leuteinizing hormone (LH) suppression.

Identification of epididymis-specific transcripts in the mouse and rat by transcriptional profiling.

As part of our efforts to identify novel contraceptive targets in the epididymis we performed transcriptional profiling on each of the 10 and 19 segments of the mouse and rat epididymidis, respectively, using Affymetrix whole genome microarrays. A total of 17 096 and 16 360 probe sets representing transcripts were identified as being expressed in the segmented mouse and rat epididymal transcriptomes, respectively. Comparison of the expressed murine transcripts against a mouse transcriptional profiling database derived from 22 other mouse tissues identified 77 transcripts that were expressed uniquely in the epididymis. The expression of these genes was further evaluated by reverse transcription polymerase chain reaction (RT-PCR) analysis of RNA from 21 mouse tissues. RT-PCR analysis confirmed epididymis-specific expression of Defensin Beta 13 and identified two additional genes with expression restricted only to the epididymis and testis. Comparison of the 16 360 expressed transcripts in the rat epididymis with data of 21 other tissues from a rat transcriptional profiling database identified 110 transcripts specific for the epididymis. Sixty-two of these transcripts were further investigated by qPCR analysis. Only Defensin 22 (E3 epididymal protein) was shown to be completely specific for the epididymis. In addition, 14 transcripts showed more than 100-fold selective expression in the epididymis. The products of these genes might play important roles in epididymal and/or sperm function and further investigation and validation as contraceptive targets are warranted. The results of the studies described in this report are available at the Mammalian Reproductive Genetics (MRG) Database (http://mrg.genetics.washington.edu/).

Gene expression during development of fetal and adult Leydig cells.

In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

Identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells.

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

The rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides.

Regional differences along the epididymis are essential for the establishment of the luminal environment required for sperm maturation. In the current study, 19 morphologically distinct segments of the rat epididymis were identified by microdissection. Total RNA was isolated from each segment and subjected to microarray analysis. Segmental analysis of epididymal gene expression identified more than 16,000 expressed qualifiers, whereas profiling of RNA from whole rat epididymis identified approximately 12,000 expressed qualifiers. Screening a panel of normal rat tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, more than 3500 qualifiers were shown to be present and differentially upregulated or downregulated by more than fourfold between any two segments. The present study complements our previous segment-dependent analysis of gene expression in the mouse epididymis and allows for comparative analyses between datasets. A total of 492 genes was shown to be present on both the MOE430 (mouse) and RAE230_2 (rat) microarrays, expressed in the epididymis of both species, and differentially expressed by more than fourfold in between segments in each species. Moreover, in-depth quantitative RT-PCR analysis of 36 members of the beta defensin gene family showed highly conserved patterns of expression along the lengths of the mouse and rat epididymides. These analyses elucidate global gene expression patterns along the length of the rat epididymis and provide a novel evaluation of conserved and nonconserved gene expression patterns in the epididymides of the two species. Furthermore, these data provide a powerful resource for the research community for future studies of biological factors that mediate sperm maturation and storage.

Gene expression profiling and its practice in drug development.

The availability of sequenced genomes of human and many experimental animals necessitated the development of new technologies and powerful computational tools that are capable of exploiting these genomic data and ask intriguing questions about complex nature of biological processes. This gave impetus for developing whole genome approaches that can produce functional information of genes in the form of expression profiles and unscramble the relationships between variation in gene expression and the resulting physiological outcome. These profiles represent genetic fingerprints or catalogue of genes that characterize the cell or tissue being studied and provide a basis from which to begin an investigation of the underlying biology. Among the most powerful and versatile tools are high-density DNA microarrays to analyze the expression patterns of large numbers of genes across different tissues or within the same tissue under a variety of experimental conditions or even between species. The wide spread use of microarray technologies is generating large sets of data that is stimulating the development of better analytical tools so that functions can be predicted for novel genes. In this review, the authors discuss how these profiles are being used at various stages of the drug discovery process and help in the identification of new drug targets, predict the function of novel genes, and understand individual variability in response to drugs.

Regulation of female fertility and identification of future contraceptive targets.

Mammalian reproduction is a complex physiological process involving a tightly regulated hypothalamic-pituitary-gonadal axis and the integration of a diverse array of molecular signals. Oral contraceptives (OCs) were introduced over 40 years ago and have evolved over the years through the discovery of new estrogens and progestins, the development of progestin-only pills and the reduction of the estrogen content in combined OCs. Despite the developments that improved the safety profile of current OCs, adverse metabolic and vascular effects caused by the estrogen component and possible neoplastic effects of OCs remain and, thus, necessitate efforts to develop newer, possibly non-steroidal and non-hormonal, contraceptives. Recent advances in our understanding of ovarian endocrinology, coupled with molecular biology and transgenic technology, have enabled identification of several factors that are functionally critical in the regulation of female fertility. Progress in the area of female reproduction is showing great promise for identifying new contraceptive drug targets. In this article, the authors review the field of female contraception with emphasis on novel targets involved in reproductive function and identified through genomics and proteomics. In addition, the usefulness of these targets for contraception purposes will be discussed.

Identification of rat cysteine-rich secretory protein 4 (Crisp4) as the ortholog to human CRISP1 and mouse Crisp4.

Cysteine-rich secretory proteins (CRISPs) are present in a diverse population of organisms and are defined by 16 conserved cysteine residues spanning a plant pathogenesis related-1 and a C-terminal cysteine-rich domain. To date, the diversification of mammalian CRISPs is evidenced by the existence of two, three, and four paralogous genes in the rat, human, and mouse, respectively. The current study identifies a third rat Crisp paralog we term Crisp4. The gene for Crisp4 is on rat chromosome 9 within 1 Mb of both the Crisp1 and Crisp2 genes. The full-length transcript for this gene was cloned from rat epididymal RNA and encodes a protein that shares 69% and 91% similarity with human CRISP1 and mouse CRISP4, respectively. Expression of rat Crisp4 is most abundant in the epididymis, with the highest levels of transcription observed in the caput and corpus epididymis. In contrast, rat CRISP4 protein is most abundant in the corpus and cauda regions of the epididymis. Rat CRISP4 protein is also present in caudal sperm extracts, appearing as a detergent-soluble form at the predicted MWR (26 kDa). Our data identify rat Crisp4 as the true ortholog to human CRISP1 and mouse Crisp4, and demonstrate its interaction with spermatozoa in the epididymis.

Segregation of micron-scale membrane sub-domains in live murine sperm.

Lipid rafts, membrane sub-domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside G(M1) localized to a micron-scale membrane sub-domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub-domain maintenance. G(M1) segregation was maintained in live sperm incubated under non-capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of G(M1) diffusion to the post-acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, G(M1) dramatically redistributed several microns from the acrosomal sub-domain to the post-acrosomal, non-raft sub-domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid-lipid interaction model for sub-domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin-1 maintained segregation of both sterols and G(M1), arguing against a role for lipid shells surrounding caveolin-1 in sub-domain maintenance. Scanning electron microscopy of sperm freeze-dried without fixation identified cytoskeletal structures at the sub-domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of G(M1), we found that disulfide-bonded proteins played a significant role in sub-domain segregation. Together, these data provide an example of membrane sub-domains extreme in terms of size and stability of lipid segregation, and implicate a protein-based membrane compartmentation mechanism.

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

The mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis.

Maturation of spermatozoa, including the acquisition of motility and the ability to undergo capacitation, occurs during transit through the dynamic environment of the epididymis. The microenvironments created along the length of the epididymal tubule are essential to the molecular modifications of spermatozoa that result in fertile gametes. The secretory and resorptive processes of the epithelial cells that line this tubule generate these microenvironments. In the current study, 10 morphologically distinct segments of the mouse epididymis were identified by microdissection. We hypothesized that the changing environments of the epididymal lumen are established by differential gene expression among these segments. RNA isolated from each of the 10 segments was analyzed by microarray analysis. More than 17,000 genes are expressed in the mouse epididymis, compared with about 12,000 genes identified from whole epididymal samples. Screening a panel of normal mouse tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, this study identified 2168 genes that are up-regulated or down-regulated by greater than 4-fold between at least two different segments. The expression patterns of these genes identify distinct patterns of segmental regulation. Using principal component analysis, we determined that the 10 segments form 6 different transcriptional units. These analyses elucidate the changes in gene expression along the length of the epididymis for 17,000 expressed transcripts and provide a powerful resource for the research community in future studies of the biological factors that mediate epididymal sperm maturation.

Analysis of the human sperm proteome.

As part of our effort to identify putative protein targets for the development of male contraceptives, we performed an in-depth proteomic analysis of human sperm by liquid chromatography and tandem mass spectrometry. Motile sperm were collected from a single fertile individual and fractionated into detergent-soluble and detergent-insoluble fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of these fractions, followed by manual cutting of the gel, yielded 35 gel sections for each fraction to include proteins across the full range of electrophoretic mobility. Proteomic analysis of these gel sections identified more than 1,760 proteins with high confidence, with 1,350 proteins identified in the soluble fraction, 719 identified in the insoluble fraction, and 309 identified in both fractions. This characterization of the human sperm proteome provides a high-resolution, physiologically relevant index of the proteins that comprise human sperm.