RUNX transcription factors are essential in maintaining epididymal epithelial differentiation.

Apart from the androgen receptor, transcription factors (TFs) that are required for the development and formation of the different segments of the epididymis have remained unknown. We identified TF families expressed in the developing epididymides, of which many showed segment specificity. From these TFs, down-regulation of runt related transcription factors (RUNXs) 1 and 2 expression coincides with epithelial regression in Dicer1 cKO mice. Concomitant deletion of both Runx1 and Runx2 in a mouse epididymal epithelial cell line affected cell morphology, adhesion and mobility in vitro. Furthermore, lack of functional RUNXs severely disturbed the formation of 3D epididymal organoid-like structures. Transcriptomic analysis of the epididymal cell organoid-like structures indicated that RUNX1 and RUNX2 are involved in the regulation of MAPK signaling, NOTCH pathway activity, and EMT-related gene expression. This suggests that RUNXs are master regulators of several essential signaling pathways, and necessary for the maintenance of proper differentiation of the epididymal epithelium.

Rationale and design of the DAPA-MI trial: Dapagliflozin in patients without diabetes mellitus with acute myocardial infarction.

BACKGROUND: Therapies that could further prevent the development of heart failure (HF) and other cardiovascular and metabolic events in patients with recent myocardial infarction (MI) represent a large and unmet medical need. METHODS: DAPA-MI is a multicenter, parallel-group, registry-based, randomized, double-blind, placebo-controlled phase 3 trial in patients without known diabetes or established HF, presenting with MI and impaired left ventricular systolic function or Q-wave MI. The trial evaluated the effect of dapagliflozin 10 mg vs placebo, given once daily in addition to standard of care therapy, on death, hospitalization for HF (HHF), and other cardiometabolic outcomes. The primary objective of the trial was to determine, using the win-ratio method, if dapagliflozin is superior to placebo by comparing the hierarchical composite outcome of death, HHF, nonfatal MI, atrial fibrillation/flutter, new onset of type 2 diabetes mellitus, HF symptoms as measured by New York Heart Association Functional Classification at last visit, and body weight decrease ≥5% at last visit. Assuming a true win-ratio of 1.20 between dapagliflozin and placebo, 4,000 patients provide a statistical power of 80% for the test of the primary composite outcome. A registry-based randomized controlled trial framework allowed for recruitment, randomization, blinding, and pragmatic data collection of baseline demographics, medications, and clinical outcomes using existing national clinical registries (in Sweden and the UK) integrated with the trial database. CONCLUSIONS: The trial explores opportunities to improve further the outcome of patients with impaired LV function after MI. The innovative trial design of DAPA-MI, incorporating national clinical registry data, has facilitated efficient patient recruitment as well as outcome ascertainment. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT04564742.

Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle.

Mammalian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation, and release of the eggs, i.e., ovulation. The steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered membrane progesterone receptor α/ß hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation of Abhd2 caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries of Abhd2 knockout (KO) females resemble polycystic ovary morphology (PCOM) with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowed Abhd2 KO females to ovulate a significantly increased number of mature and fertile eggs in comparison with their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in non-genomic steroid regulation of the female reproductive cycle.

The epithelial potassium channel Kir7.1 is stimulated by progesterone.

The choroid plexus (CP) epithelium secretes cerebrospinal fluid and plays an important role in healthy homeostasis of the brain. CP function can be influenced by sex steroid hormones; however, the precise molecular mechanism of such regulation is not well understood. Here, using whole-cell patch-clamp recordings from male and female murine CP cells, we show that application of progesterone resulted in specific and strong potentiation of the inwardly rectifying potassium channel Kir7.1, an essential protein that is expressed in CP and is required for survival. The potentiation was progesterone specific and independent of other known progesterone receptors expressed in CP. This effect was recapitulated with recombinant Kir7.1, as well as with endogenous Kir7.1 expressed in the retinal pigment epithelium. Current-clamp studies further showed a progesterone-induced hyperpolarization of CP cells. Our results provide evidence of a progesterone-driven control of tissues in which Kir7.1 is present.

Choroid plexus epithelial cells as a model to study nongenomic steroid signaling and its effect on ion channel function.

The choroid plexus (CP) is an epithelial tissue primarily responsible for the secretion of the cerebrospinal fluid (CSF). Choroid plexuses are found in each of the four brain ventricles: two laterals, third and fourth. They ensure continuous production of CSF to provide nutrients, remove waste products and provide a mechanical buffer to protect the brain. Tight junctions in the CP epithelium form a barrier between the blood plasma and the CSF, which allow channels and transporters in the CP to establish a highly regulated concentration gradient of ions between the two fluids, thereby controlling the composition of CSF. CP plays an important part in healthy brain homeostasis, as its failure to maintain adequate CSF perfusion is implicated in Alzheimer's disease and traumatic brain injury. And yet, the physiology of CP and the mechanism of its age-related functional decline is one of the most understudied areas of neurobiology. Here, we describe a protocol to isolate and identify individual choroid plexus epithelial cells (CPEC) from murine brain for whole-cell patch-clamp recordings and ion channel identification. Using the recording from the inwardly rectifying potassium channel Kir7.1 and TRPM3 that are abundant in CP, we demonstrate a technique to study the regulators of ion channels in the choroid plexus.

The impact of epididymal proteins on sperm function.

The epididymis is necessary for post-testicular sperm maturation as it provides the milieu required for spermatozoa to gain the ability for progressive movement and fertilization. In the epididymis the sperm protein, lipid and small RNA content are heavily modified due to interaction with luminal proteins secreted by the epididymal epithelium and extracellular vesicles, epididymosomes. This review focuses on epididymal proteins demonstrated to have an effect on sperm functions, such as motility, capacitation, acrosome reaction, sperm-zona pellucida binding and sperm-egg binding, as well as on embryonic development.

Role of Dicer1-Dependent Factors in the Paracrine Regulation of Epididymal Gene Expression.

Dicer1 is an endoribonuclease involved in the biogenesis of functional molecules such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs). These small non-coding RNAs are important regulators of post-transcriptional gene expression and participate in the control of male fertility. With the knowledge that 1) Dicer1-dependent factors are required for proper sperm maturation in the epididymis, and that 2) miRNAs are potent mediators of intercellular communication in most biological systems, we investigated the role of Dicer1-dependent factors produced by the proximal epididymis (initial segment/caput)- including miRNAs- on the regulation of epididymal gene expression in the distal epididymis regions (i.e. corpus and cauda). To this end, we performed comparative microarray and ANOVA analyses on control vs. Defb41iCre/wt;Dicer1fl/fl mice in which functional Dicer1 is absent from the principal cells of the proximal epididymis. We identified 35 and 33 transcripts that displayed significant expression level changes in the corpus and cauda regions (Fold change > 2 or < -2; p < 0.002), respectively. Among these transcripts, Zn-alpha 2-glycoprotein (Azgp1) encodes for a sperm equatorial protein whose expression in the epididymis of Dicer1 cKO mice is significantly increased compared to controls. In addition, 154 miRNAs, including miR-210, miR-672, miR-191 and miR-204, showed significantly impaired biogenesis in the absence of Dicer1 from the principal cells of the proximal epididymis (Fold change > 2 or < -2; p < 0.01). These miRNAs are secreted via extracellular vesicles (EVs) derived from the DC2 epididymal principal cell line, and their expression correlates with target transcripts involved in distinct biological pathways, as evidenced by in silico analysis. Albeit correlative and based on in silico approach, our study proposes that Dicer1-dependent factors trigger- directly or not-significant genes expression changes in distinct regions of this organ. The paracrine control of functions important to post-testicular sperm maturation by Dicer1-dependent factors may open new avenues for the identification of molecular targets important to male fertility control.

Segment-specific regulation of epididymal gene expression.

The epididymis is necessary for post-testicular sperm maturation. During their epididymal transit, spermatozoa gain ability for progressive movement and fertilization. The epididymis is composed of several segments that have distinct gene expression profiles that enable the establishment of the changing luminal environment required for sperm maturation. The epididymal gene expression is regulated by endocrine, lumicrine, and paracrine factors in a segment-specific manner. Thus, in addition to its importance for male fertility, the epididymis is a valuable model tissue for studying the regulation of gene expression. This review concentrates on recent advances in understanding the androgen, small RNA, and epigenetically mediated regulation of segment-specific gene expression in the epididymis.

Targeted inactivation of the mouse epididymal beta-defensin 41 alters sperm flagellar beat pattern and zona pellucida binding.

During epididymal maturation, sperm acquire the ability to swim progressively by interacting with proteins secreted by the epididymal epithelium. Beta-defensin proteins, expressed in the epididymis, continue to regulate sperm motility during capacitation and hyperactivation in the female reproductive tract. We characterized the mouse beta-defensin 41 (DEFB41), by generating a mouse model with iCre recombinase inserted into the first exon of the gene. The homozygous Defb41(iCre/iCre) knock-in mice lacked Defb41 expression and displayed iCre recombinase activity in the principal cells of the proximal epididymis. Heterozygous Defb41(iCre/+) mice can be used to generate epididymis specific conditional knock-out mouse models. Homozygous Defb41(iCre/iCre) sperm displayed a defect in sperm motility with the flagella primarily bending in the pro-hook conformation while capacitated wild-type sperm more often displayed the anti-hook conformation. This led to a reduced straight line motility of Defb41(iCre/iCre) sperm and weaker binding to the oocyte. Thus, DEFB41 is required for proper sperm maturation.

The role of Dicer1 in the male reproductive tract.

Dicer1 is an RNase III enzyme necessary for microRNA (miRNA) biogenesis, as it cleaves pre-miRNAs into mature miRNAs. miRNAs are important regulators of gene expression. In recent years, several miRNA-independent roles of Dicer1 have been identified. They include the production of endogenous small interfering RNAs, detoxifying retrotransposon-derived transcripts, and binding to new targets; messenger RNAs and long noncoding RNAs. Further, in this review, the functional significance of Dicer1 in the male reproductive tract is discussed. Conditional Dicer1 knock-out mouse models have demonstrated a requisite role for Dicer in male fertility. Deletion of Dicer1 from somatic or germ cells in the testis cause spermatogenic problems rendering male mice infertile. The lack of Dicer1 in the proximal epididymis causes dedifferentiation of the epithelium, with unbalanced sex steroid receptor expression, defects in epithelial lipid homeostasis, and subsequent male infertility. In addition, Dicer1 ablation from the prostate leads to increased apoptosis of the differentiated luminal cells, followed by epithelial hypotrophy of the ventral prostate. However, further studies are needed to clarify which functions of Dicer1 are responsible for the observed phenotypes in the male reproductive tract.

Imbalanced lipid homeostasis in the conditional Dicer1 knockout mouse epididymis causes instability of the sperm membrane.

During epididymal sperm maturation, the lipid content of the sperm membrane is modified, which facilitates sperm motility and fertility. However, little is known about the mechanisms regulating the maturation process. By generating a conditional knockout (cKO) of Dicer1 in the proximal part of the mouse epididymis, we studied the role of RNA interference in epididymal functions. The Dicer1 cKO epididymis displayed an altered lipid homeostasis associated with a 0.6-fold reduction in the expression of the gene elongation of very long chain fatty acids-like 2, an enzyme needed for production of long-chain polyunsaturated fatty acids (PUFAs). Furthermore, the expression of several factors involved in cholesterol synthesis was up-regulated. Accordingly, the Dicer1 cKO sperm membrane showed a 0.7-fold decrease in long-chain PUFAs, whereas the amount of cholesterol in acrosome-reacted sperm displayed a 1.7-fold increase. The increased cholesterol:PUFA ratio of the sperm membrane caused breakage of the neck and acrosome region and immotility of sperm. Dicer1 cKO mice sperm also displayed reduced ability to bind to and fertilize the oocyte in vitro. This study thus shows that Dicer1 is critical for lipid synthesis in the epididymis, which directly affects sperm membrane integrity and male fertility.

Loss of Bmyc results in increased apoptosis associated with upregulation of Myc expression in juvenile murine testis.

Bmyc is a member of the Myc family of transcriptional regulators in the mouse and the rat. It is predominantly expressed in hormonally controlled tissues, with highest level of expression in the epididymis. The BMYC protein has been shown to function as a transcription factor in vitro and to inhibit MYC. To study the significance of BMYC in vivo, a Bmyc knockout (KO) mouse model was generated by homologous recombination. The KO mice were viable and fertile and did not display gross morphological or histological changes compared to the WT mice. However, the testes and the epididymides of the KO mice were smaller than those of the WT mice. Correspondingly, a tendency for a lower sperm concentration in the cauda epididymides of the KO mice was detected. The testosterone produced/testis was significantly reduced, and accordingly, the LH levels were increased in the KO mice. Also, the expression levels of Myc and several of its target genes were elevated in the testes of prepubertal KO mice, whereas no differences in gene expression levels were detected in adult mice. Associated with the increased Myc expression, more apoptotic spermatogenic cells were detected in the seminiferous tubules of the KO mice. In conclusion, our data suggest that Bmyc is a regulator of Myc in vivo and that overexpression of Myc in the developing testis leads to increased apoptosis of spermatogenic cells.

Dicer1 ablation in the mouse epididymis causes dedifferentiation of the epithelium and imbalance in sex steroid signaling.

BACKGROUND: The postnatal development of the epididymis is a complex process that results in a highly differentiated epithelium, divided into several segments. Recent studies indicate a role for RNA interference (RNAi) in the development of the epididymis, however, the actual requirement for RNAi has remained elusive. Here, we present the first evidence of a direct need for RNAi in the differentiation of the epididymal epithelium. METHODOLOGY/PRINCIPAL FINDINGS: By utilizing the Cre-LoxP system we have generated a conditional knock-out of Dicer1 in the two most proximal segments of the mouse epididymis. Recombination of Dicer1, catalyzed by Defb41(iCre/wt), took place before puberty, starting from 12 days postpartum. Shortly thereafter, downregulation of the expression of two genes specific for the most proximal epididymis (lipocalin 8 and cystatin 8) was observed. Following this, segment development continued until week 5 at which age the epithelium started to regress back to an undifferentiated state. The dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was downregulated, indicating imbalanced sex steroid signaling. CONCLUSIONS/SIGNIFICANCE: At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways essential for maintaining epithelial cell differentiation.

Loss of cysteine-rich secretory protein 4 (Crisp4) leads to deficiency in sperm-zona pellucida interaction in mice.

Mammalian sperm gain their ability to fertilize the egg during transit through the epididymis and by interacting with proteins secreted by the epididymal epithelial cells. Certain members of the CRISP (cysteine-rich secretory protein) family form the major protein constituent of the luminal fluid in the mammalian epididymis. CRISP4 is the newest member of the CRISP family expressed predominantly in the epididymis. Its structure and expression pattern suggest a role in sperm maturation and/or sperm-egg interaction. To study the relevance of CRISP4 in reproduction, we have generated a Crisp4 iCre knock-in mouse model through insertion of the iCre recombinase coding cDNA into the Crisp4 locus. This allows using the mouse line both as a Crisp4 deficient model and as an epididymis-specific iCre-expressing mouse line applicable for the generation of conditional, epididymis-specific knockout mice. We show that the loss of CRISP4 leads to a deficiency of the spermatozoa to undergo progesterone-induced acrosome reaction and to a decreased fertilizing ability of the sperm in the in vitro fertilization conditions, although the mice remain fully fertile in normal mating. However, removal of the egg zona pellucida returned the fertilization potential of the CRISP4-deficient spermatozoa, and accordingly we detected a reduced number of Crisp4-deficient spermatozoa bound to oocytes as compared with the wild-type spermatozoa. We also demonstrate that iCre recombinase is expressed in a pattern similar to endogenous Crisp4 and is able to initiate the recombination event with its target sequences in vivo.

Members of the murine Pate family are predominantly expressed in the epididymis in a segment-specific fashion and regulated by androgens and other testicular factors.

BACKGROUND: Spermatozoa leaving the testis are not able to fertilize the egg in vivo. They must undergo further maturation in the epididymis. Proteins secreted to the epididymal lumen by the epithelial cells interact with the spermatozoa and enable these maturational changes, and are responsible for proper storage conditions before ejaculation. The present study was carried out in order to characterize the expression of a novel Pate (prostate and testis expression) gene family, coding for secreted cysteine-rich proteins, in the epididymis. METHODS: Murine genome databases were searched and sequence comparisons were performed to identify members of the Pate gene family, and their expression profiles in several mouse tissues were characterized by RT-PCR. Alternate transcripts were identified by RT-PCR, sequencing and Northern hybridization. Also, to study the regulation of expression of Pate family genes by the testis, quantitative (q) RT-PCR analyses were performed to compare gene expression levels in the epididymides of intact mice, gonadectomized mice, and gonadectomized mice under testosterone replacement treatment. RESULTS: A revised family tree of Pate genes is presented, including a previously uncharacterized Pate gene named Pate-X, and the data revealed that Acrv1 and Sslp1 should also be considered as members of the Pate family. Alternate splicing was observed for Pate-X, Pate-C and Pate-M. All the Pate genes studied are predominantly expressed in the epididymis, whereas expression in the testis and prostate is notably lower. Loss of androgens and/or testicular luminal factors was observed to affect the epididymal expression of several Pate genes. CONCLUSIONS: We have characterized a gene cluster consisting of at least 14 expressed Pate gene members, including Acrv1, Sslp1 and a previously uncharacterized gene which we named Pate-X. The genes code for putatively secreted, cysteine-rich proteins with a TFP/Ly-6/uPAR domain. Members of the Pate gene cluster characterized are predominantly expressed in the murine epididymis, not in the testis or prostate, and are regulated by testicular factors. Similar proteins are present in venoms of several reptiles, and they are thought to mediate their effects by regulating certain ion channels, and are thus expected to have a clinical relevance in sperm maturation and epididymal infections.

The Drosophila DPP signal is produced by cleavage of its proprotein at evolutionary diversified furin-recognition sites.

Maturation of bone morphogenetic proteins (BMPs) requires cleavage of their precursor proteins by furin-type proprotein convertases. Here, we find that cleavage sites of the BMP2/4/decapentaplegic (DPP) subfamily have been evolutionary diversified and can be categorized into 4 different types. Cnidaria BMP2/4/DPP is considered to be a prototype containing only 1 furin site. Bilateria BMP2/4/DPP acquired an additional cleavage site with either the combination of minimal-optimal or optimal-optimal furin sites. DPPs belonging to Diptera, such as Drosophila and mosquito, and Lepidoptera of silkworm contain a third cleavage site between the 2 optimal furin sites. We studied how the 3 furin sites (FSI-III) of Drosophila DPP coordinate maturation of ligands and contribute to signals in vivo. Combining mutational analysis of furin-recognition sites and RNAi experiments, we found that the Drosophila DPP precursor is initially cleaved at an upstream furin-recognition site (FSII), with consequent cleavages at 2 furin sites (FSI and FSIII). Both Dfurin1 and Dfurin2 are involved in the processing of DPP proproteins. Biochemical and genetic analyses using cleavage mutants of DPP suggest the first cleavage at FSII to be critical and sufficient for long-range DPP signaling. Our data suggest that the Drosophila DPP precursor is cleaved in a different manner from vertebrate BMP4 even though they are functional orthologs. This indicates that the furin-cleavage sites in BMP2/4/DPP precursors are tolerant to mutations acquired through evolution and have adapted to different systems in diversified species.