SOX9 Modulates the Transformation of Gastric Stem Cells Through Biased Symmetric Cell Division.

BACKGROUND & AIMS: Transformation of stem/progenitor cells has been associated with tumorigenesis in multiple tissues, but stem cells in the stomach have been hard to localize. We therefore aimed to use a combination of several markers to better target oncogenes to gastric stem cells and understand their behavior in the initial stages of gastric tumorigenesis. METHODS: Mouse models of gastric metaplasia and cancer by targeting stem/progenitor cells were generated and analyzed with techniques including reanalysis of single-cell RNA sequencing and immunostaining. Gastric cancer cell organoids were genetically manipulated with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for functional studies. Cell division was determined by bromodeoxyuridine-chasing assay and the assessment of the orientation of the mitotic spindles. Gastric tissues from patients were examined by histopathology and immunostaining. RESULTS: Oncogenic insults lead to expansion of SOX9+ progenitor cells in the mouse stomach. Genetic lineage tracing and organoid culture studies show that SOX9+ gastric epithelial cells overlap with SOX2+ progenitors and include stem cells that can self-renew and differentiate to generate all gastric epithelial cells. Moreover, oncogenic targeting of SOX9+SOX2+ cells leads to invasive gastric cancer in our novel mouse model (Sox2-CreERT;Sox9-loxp(66)-rtTA-T2A-Flpo-IRES-loxp(71);Kras(Frt-STOP-Frt-G12D);P53R172H), which combines Cre-loxp and Flippase-Frt genetic recombination systems. Sox9 deletion impedes the expansion of gastric progenitor cells and blocks neoplasia after Kras activation. Although Sox9 is not required for maintaining tissue homeostasis where asymmetric division predominates, loss of Sox9 in the setting of Kras activation leads to reduced symmetric cell division and effectively attenuates the Kras-dependent expansion of stem/progenitor cells. Similarly, Sox9 deletion in gastric cancer organoids reduces symmetric cell division, organoid number, and organoid size. In patients with gastric cancer, high levels of SOX9 are associated with recurrence and poor prognosis. CONCLUSION: SOX9 marks gastric stem cells and modulates biased symmetric cell division, which appears to be required for the malignant transformation of gastric stem cells.

Investigation of selective retinoic acid receptor alpha antagonist ER-50891 and related analogs for male contraception.

Retinoic acid receptor alpha (RARα) antagonist ER-50891 and 15 analogs were prepared and tested in vitro for potency and selectivity at RARα, RARß, and RARγ using transactivation assays. Minor modifications to the parent molecule such as the introduction of a C4 tolyl group in place of the C4 phenyl group on the quinoline moiety slightly increased the RARα selectivity but larger substituents significantly decreased the potency. Replacement of the pyrrole moiety of ER-50891 with triazole, amides, or a double bond produced inactive compounds. ER-50891 was found to be stable in male mouse liver microsomes and was tested in male mice to assess its effects on spermatogenesis. Characteristic, albeit modest and transient, effects on spermatogenesis were observed.

Filamentous actin disorganization and absence of apical ectoplasmic specialization disassembly during spermiation upon interference with retinoid signaling†.

Spermiation is a multiple-step process involving profound cellular changes in both spermatids and Sertoli cells. We have observed spermiation defects, including abnormalities in spermatid orientation, translocation and release, in mice deficient in the retinoic acid receptor alpha (RARA) and upon treatment with a pan-RAR antagonist. To elucidate the role of retinoid signaling in regulating spermiation, we first characterized the time course of appearance of spermiogenic defects in response to treatment with the pan-RAR antagonist. The results revealed that defects in spermiation are indeed among the earliest abnormalities in spermatogenesis observed upon inhibition of retinoid signaling. Using fluorescent dye-conjugated phalloidin to label the ectoplasmic specialization (ES), we showed for the first time that these defects involved improper formation of filamentous actin (F-actin) bundles in step 8-9 spermatids and a failure of the actin-surrounded spermatids to move apically to the lumen and to disassemble the ES. The aberrant F-actin organization is associated with diminished nectin-3 expression in both RARA-deficient and pan-RAR antagonist-treated testes. An abnormal localization of both tyrosinated and detyrosinated tubulins was also observed during spermatid translocation in the seminiferous epithelium in drug-treated testes. These results highlight a crucial role of RAR receptor-mediated retinoid signaling in regulating microtubules and actin dynamics in the cytoskeleton rearrangements, required for proper spermiation. This is critical to understand in light of ongoing efforts to inhibit retinoid signaling as a novel approach for male contraception and may reveal spermiation components that could also be considered as new targets for male contraception.

Retinoic acid receptor antagonists for male contraception: current status†.

Retinoic acid receptor alpha (RARA), a nuclear receptor protein, has been validated as a target for male contraception by gene knockout studies and also pharmacologically using a pan-retinoic acid receptor antagonist. Retinoic acid receptor alpha activity is indispensable for the spermatogenic process, and therefore its antagonists have potential as male contraceptive agents. This review discusses the effects of systematic dosing regimen modifications of the orally bioavailable and reversible pan-antagonist BMS-189453 as well as studies with the alpha-selective antagonists BMS-189532 and BMS-189614 in a murine model. We also provide an overview of structure-activity studies of retinoic acid receptor alpha antagonists that provide insight for the design of novel alpha-selective ligands.

Mammalian E-type cyclins control chromosome pairing, telomere stability and CDK2 localization in male meiosis.

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.

Strategies for developing retinoic acid receptor alpha-selective antagonists as novel agents for male contraception.

Reported here are the synthesis and in vitro evaluation of a series of 26 retinoic acid analogs based on dihydronaphthalene and chromene scaffolds using a transactivation assay. Chromene amide analog 21 was the most potent and selective retinoic acid receptor α antagonist identified from this series. In vitro evaluation indicated that 21 has favorable physicochemical properties and a favorable pharmacokinetic PK profile in vivo with significant oral bioavailability, metabolic stability, and testes exposure. Compound 21 was evaluated for its effects on spermatogenesis and disruption of fertility in a mouse model. Oral administration of compound 21 at low doses showed reproducibly characteristic albeit modest effects on spermatogenesis, but no effects on fertility were observed in mating studies. The inhibition of spermatogenesis could not be enhanced by raising the dose and lengthening the duration of dosing. Thus, 21 may not be a good candidate to pursue further for effects on male fertility.

Cyclin A2 is essential for mouse gonocyte maturation.

In mammals, male gonocytes are derived from primordial germ cells during embryogenesis, enter a period of mitotic proliferation, and then become quiescent until birth. After birth, the gonocytes proliferate and migrate from the center of testicular cord toward the basement membrane to form the pool of spermatogonial stem cells (SSCs) and establish the SSC niche architecture. However, the molecular mechanisms underlying gonocyte proliferation, migration and differentiation are largely unknown. Cyclin A2 is a key component of the cell cycle and required for cell proliferation. Here, we show that cyclin A2 is required in mouse male gonocyte development and the establishment of spermatogenesis in the neonatal testis. Loss of cyclin A2 function in embryonic gonocytes by targeted gene disruption affected the regulation of the male gonocytes to SSC transition, resulting in the disruption of SSC pool formation, imbalance between SSC self-renewal and differentiation, and severely abnormal spermatogenesis in the adult testis.

Prolonged Oral Administration of a Pan-Retinoic Acid Receptor Antagonist Inhibits Spermatogenesis in Mice With a Rapid Recovery and Changes in the Expression of Influx and Efflux Transporters.

We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

Apoptosis in male germ cells in response to cyclin A1-deficiency and cell cycle arrest.

Male mice homozygous for a mutated allele of the cyclin A1 gene (Ccna1) are sterile due to a block in cell cycle progression before the first meiotic division. Meiosis arrest in Ccna1(-/-) spermatocytes is associated with desynapsis abnormalities, lowered MPF activity, and apoptosis as evidenced by TUNEL-positive staining. With time, adult testicular tubules exhibit severe degeneration: some tubules in the older animals are almost devoid of germ cells at various stages of spermatogenesis. The mechanisms by which the cells sense the cell cycle arrest and the regulation of the decision to undergo cell death are under investigation.

Pharmacological activity of retinoic acid receptor alpha-selective antagonists in vitro and in vivo.

Oral administration of a retinoic acid receptor (RAR) pan-antagonist reversibly inhibits spermatogenesis. Given the importance of RARα in regulating spermatogenesis, we identified two RARα-selective antagonists by transactivation and transactivation competition assays and asked whether they effectively inhibit spermatogenesis. Although these two antagonists were potent in vitro, they displayed poor in vivo activity in mice when administered orally. Testicular weights were normal and morphological analysis revealed normal spermatid alignment and sperm release. In vitro drug property analyses were performed with one of these antagonists and compared with the pan-antagonist. We showed that the discrepancies may be explained by several factors, including high plasma protein binding, faster hepatic metabolism relative to the pan-antagonist, and only moderate permeability. The conclusion of poor oral bioavailability was supported by more pronounced defects in mice when the antagonist was administered intravenously versus intraperitoneally. These results are crucial for designing new RARα-selective antagonists for pharmaceutical application.

Oral administration of a retinoic Acid receptor antagonist reversibly inhibits spermatogenesis in mice.

Here we investigated a pharmacological approach to inhibit spermatogenesis in the mouse model by manipulating retinoid signaling using low doses of the pan-retinoic acid receptor (RAR) antagonist BMS-189453. Spermatogenesis was disrupted, with a failure of spermatid alignment and sperm release and loss of germ cells into lumen, abnormalities that resembled those in vitamin A-deficient and RARα-knockout testes. Importantly, the induced sterility was reversible. Enhanced efficacy and a lengthened infertility period with full recovery of spermatogenesis were observed using systematically modified dosing regimens. Hematology, serum chemistry, and hormonal and pathological evaluations revealed no detectable abnormalities or adverse side effects except the distinct testicular pathology. Our results suggest that testes are exquisitely sensitive to disruption of retinoid signaling and that RAR antagonists may represent new lead molecules in developing nonsteroidal male contraceptives.

Aberrant distribution of junctional complex components in retinoic acid receptor alpha-deficient mice.

Retinoic acid receptor alpha (RARalpha)-deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell-cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli-cell barrier in the tubules of RARalpha-deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)-responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO-1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin-40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RARalpha-deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RARalpha-deficient testes may correlate with a disrupted cyclic expression of RA-responsive structural components, including vimentin, a downregulation of connexin-40 in spermatogenic cells, and delayed assembly of ZO-1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites.

Expression of retinoic acid receptor alpha in the germline is essential for proper cellular association and spermiogenesis during spermatogenesis.

Signaling through vitamin A metabolites is indispensable for spermatogenesis, and disruption of retinoic acid receptor alpha (RARalpha) function resulted in male sterility and aberrant spermatogenesis, which resembled vitamin A deficiency. Here we investigated the lineage- and cell-specific role of RARalpha-mediated signaling during spermatogenesis using germ-cell transplantation and genetically manipulated mouse models. We demonstrated that RARalpha-deficient germ-cell stem cells were able to repopulate germ-cell-depleted wild-type testes and initiate spermatogenesis; however, improper cellular associations and abnormal sperm formation were observed. We further generated RARalpha-deficient mice that expressed RARalpha-EGFP fusion protein uniquely in haploid germ cells. Strikingly, spermatid orientation, alignment and release, as well as sperm morphology, were normal and there was a partial rescue of sterility. These data provide the first direct evidence for a distinct requirement of RARalpha-mediated retinoid signaling specifically in germ cells.

The conserved transcriptome in human and rodent male gametogenesis.

We report a cross-species expression profiling analysis of the human, mouse, and rat male meiotic transcriptional program, using enriched germ cell populations, whole gonads, and high-density oligonucleotide microarrays (GeneChips). Among 35% of the protein-coding genes present in rodent and human genomes that were found to be differentially expressed between germ cells and somatic controls, a key group of 357 conserved core loci was identified that displays highly similar meiotic and postmeiotic patterns of transcriptional induction across all three species. Genes known to be important for sexual reproduction are significantly enriched among differentially expressed core loci and a smaller group of conserved genes not detected in 17 nontesticular somatic tissues, correlating transcriptional activation and essential function in the male germ line. Some genes implicated in the etiology of cancer are found to be strongly transcribed in testis, suggesting that these genes may play unexpected roles in sexual reproduction. Expression profiling data further identified numerous conserved genes of biological and clinical interest previously unassociated with the mammalian male germ line.

Male sterility in mice lacking retinoic acid receptor alpha involves specific abnormalities in spermiogenesis.

The severe degeneration of the germinal epithelium and subsequent male sterility observed in mice null for the retinoic acid receptor alpha (RARalpha) gene suggested its critical role in spermatogenesis, although the etiology and progression of these abnormalities remain to be determined. Previous studies have revealed that elongated spermatids in RARalpha(-/-) testes were improperly aligned at the tubular lumen and did not undergo spermiation at stage VIII(*). We now report a distinctive failure of step 8-9 spermatids to orient properly with regard to the basal aspect of Sertoli cells, resulting in stage VIII(*)-IX(*) tubules with randomly oriented spermatids. By in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL), we noted that elongating spermatids frequently underwent apoptosis. Immunohistochemical analysis revealed that while activated caspase-3, the primary effector caspase in the apoptotic cell death machinery, was detected in the nuclei of primary spermatocytes in the first wave of spermatogenesis and occasionally in spermatogonia of both normal and mutant testes, it was not involved in the death of elongating spermatids in RARalpha(-/-) testes. Thus, sterility in RARalpha(-/-) males was associated with specific defects in spermiogenesis, which may correlate with a failure in both spermatid release and spermatid orientation to the basal aspect of Sertoli cells at stage VIII(*) in young adult RARalpha(-/-) testis. Further, the resulting apoptosis in elongating spermatids appears to involve pathways other than that mediated by activated caspase-3.

Primary spermatocyte-specific Cre recombinase activity in transgenic mice.

We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene promoter. Sycp1Cre mice were crossed with the ROSA26 reporter line R26R, to monitor the male germ cell stage-specificity of Cre activity as well as to verify that Cre was not active previously during development of other tissues. X-gal staining detected Cre-mediated recombination only in testis. Detailed histological examination indicated that weak Cre-mediated recombination occurred as early as in zygotene spermatocytes at stage XI of the cycle of the seminiferous epithelium. Robust expression of X-gal was detected in early to mid-late spermatocytes at stages V-VIII. We conclude that this transgenic line is a powerful tool for deleting genes of interest specifically during male meiosis.

Retinoic acid receptor alpha is required for synchronization of spermatogenic cycles and its absence results in progressive breakdown of the spermatogenic process.

Targeted mutagenesis of the retinoic acid receptor alpha (RAR alpha) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RAR alpha(-/-) testes, there was an increase in degenerating pachytene spermatocytes and a temporary developmental arrest in step 8-9 spermatids in the first wave of spermatogenesis, a delay in the onset of the second wave, and a temporary arrest in preleptotene to leptotene spermatocytes in the first, second, and third waves. A striking aspect of the mutant phenotype was the failure of spermatids to align at the tubular lumen at stage VIII. Furthermore, there were missing or decreased numbers of the predicted cell types in tubules, and they exhibited a profound asynchrony of mixed spermatogenic cell types. In vivo bromodeoxyuridine labeling revealed a significant decrease in germ cell proliferation in both juvenile and adult RAR alpha(-/-) testes and confirmed the arrest at step 8-9 spermatids. Retinoid signaling through RAR alpha, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations.

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis.

Rbm is a male infertility gene located on the Y chromosome that is expressed in the testis. To investigate the specific events of spermatogenesis in which Rbm plays a role, the precise pattern of expression of Rbm in the mouse testis was determined. An antibody was generated against the Rbm protein and used to detect a single specific band of 43 kDa in size in mouse testicular lysates. In situ hybridization, immunoblot and immunohistochemistry analyses together indicated that Rbm was expressed in spermatogonia, preleptotene spermatocytes, late leptotene to early pachytene spermatocytes but not in mid-pachytene spermatocytes or subsequent stages of differentiation, including haploid germ cells. These observations suggest that Rbm functions in early but not later stages of male germ cell development.