Three-Dimensional Cell Culture of Epididymal Basal Cells and Organoids: A Novel Tool for Toxicology.

Spermatozoa are formed in the testis but must transit through the epididymis to acquire motility and the ability to fertilize. The epididymis is a single convoluted tubule comprising several anatomically and physiologically distinct regions. The pseudostratified epithelium consists of multiple cell types, including principal cells, clear cells, narrow cells, and apical cells, that line the lumen of the epididymis. Basal cells are present at the base of the epithelium, and halo cells, which includes macrophages/monocytes, mononuclear phagocytes, and T lymphocytes, are also present in the epithelium. Several aspects of this complex spermatozoan maturation process are well established, but a great deal remains poorly understood. Given that dysfunction of the epididymis has been associated with male infertility, in vitro tools to study epididymal function and epididymal sperm maturation are required. Our lab and others have previously developed human, rat, and mouse epithelial principal cell lines, which have been used to address certain questions, such as about the regulation of junctional proteins in the epididymis, as well as the toxicity of nonylphenols. Given that the epididymal epithelium comprises multiple cell types, however, a 3D in vitro model provides a more comprehensive and realistic tool that can be used to study and elucidate the multiple aspects of epididymal function. The purpose of this article is to provide detailed information regarding the preparation, maintenance, passaging, and immunofluorescent staining of rat epididymal organoids derived from adult basal cells, which we have demonstrated to be a type of adult stem cell in the rat epididymis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of epididymal cells Basic Protocol 2: Magnetic activated cell sorting and isolation of basal cells Basic Protocol 3: Preparation and culture of epididymal basal cell organoids Basic Protocol 4: Passage of epididymal basal cell organoids Basic Protocol 5: Freezing and thawing of epididymal basal cell organoids Basic Protocol 6: Immunofluorescent staining of epididymal basal cell organoids.

Differential gene expression and hallmarks of stemness in epithelial cells of the developing rat epididymis.

Epididymal development can be subdivided into three phases: undifferentiated, a period of differentiation, and expansion. The objectives of this study were (1) to assess gene expression profiles in epididymides, (2) predict signaling pathways, and (3) develop a novel 3D cell culture method to assess the regulation of epididymal development in vitro. Microarray analyses indicate that the largest changes in differential gene expression occurred between the 7- to 18-day period, in which 1452 genes were differentially expressed, while 671 differentially expressed genes were noted between days 18 and 28, and there were 560 differentially expressed genes between days 28 and 60. Multiple signaling pathways were predicted at different phases of development. Pathway associations indicated that in epididymides of 7- to 18-day old rats, there was a significant association of regulated genes implicated in stem cells, estrogens, thyroid hormones, and kidney development, while androgen- and estrogen-related pathways were enriched at other phases of development. Organoids were derived from CD49f + columnar cells from 7-day old rats, while no organoids developed from CD49f- cells. Cells cultured in an epididymal basal cell organoid medium versus a commercial kidney differentiation medium supplemented with DHT revealed that irrespective of the culture medium, cells within differentiating organoids expressed p63, AQP9, and V-ATPase after 14 days of culture. The commercial kidney medium resulted in an increase in the number of organoids positive for p63, AQP9, and V-ATPase. Together, these data indicate that columnar cells represent an epididymal stem/progenitor cell population.

Parent mental health and family functioning following diagnosis of CHD: a research agenda and recommendations from the Cardiac Neurodevelopmental Outcome Collaborative.

Diagnosis of CHD substantially affects parent mental health and family functioning, thereby influencing child neurodevelopmental and psychosocial outcomes. Recognition of the need to proactively support parent mental health and family functioning following cardiac diagnosis to promote psychosocial adaptation has increased substantially over recent years. However, significant gaps in knowledge remain and families continue to report critical unmet psychosocial needs. The Parent Mental Health and Family Functioning Working Group of the Cardiac Neurodevelopmental Outcome Collaborative was formed in 2018 through support from an R13 grant from the National Heart, Lung, and Blood Institute to identify significant knowledge gaps related to parent mental health and family functioning, as well as critical questions that must be answered to further knowledge, policy, care, and outcomes. Conceptually driven investigations are needed to identify parent mental health and family functioning factors with the strongest influence on child outcomes, to obtain a deeper understanding of the biomarkers associated with these factors, and to better understand how parent mental health and family functioning influence child outcomes over time. Investigations are also needed to develop, test, and implement sustainable models of mental health screening and assessment, as well as effective interventions to optimise parent mental health and family functioning to promote psychosocial adaptation. The critical questions and investigations outlined in this paper provide a roadmap for future research to close gaps in knowledge, improve care, and promote positive outcomes for families of children with CHD.

Uptake and impact of journaling program on wellbeing of NICU parents.

OBJECTIVE: This pilot study evaluated a brief parent journaling program in the neonatal intensive care unit (NICU). STUDY DESIGN: Hundred NICU parents were randomized to a control group (no journal) or an intervention group (journal provided). Parents reported pre- and post-intervention anxiety and depression symptoms using the hospital anxiety and depression scale (HADS) and qualitative journal use data. The analysis included Student's paired two-tailed t-test and two-way ANOVA. This study was registered with clinicaltrials.gov on April 1, 2020, NCT04331925. RESULT: At baseline, clinically significant anxiety was more prevalent than depression (66% vs. 23%). Post-intervention scores were best predicted by baseline scores. Relative to controls, intervention group parents experienced a decrease in anxiety from baseline (t = -1.983, p = 0.056). The same effect was not seen for depression. Most intervention group parents used the journal and provided positive feedback. CONCLUSION: Journal use rates and positive feedback support the acceptability of a NICU journaling program.

Improved Nutrition Status in Patients With Advanced Heart Failure Implanted With a Left Ventricular Assist Device.

BACKGROUND: Left ventricular assist device (LVAD) implantation for advanced heart failure is known to improve survival, functional capacity, and quality of life. Most patients implanted with LVADs suffer from moderate to severe malnutrition and deconditioning due to their advanced disease. The Mini Nutritional Assessment (MNA) and the short form of the survey (MNA-SF) are 2 well-validated clinical tools, previously used to assess patient nutrition status in numerous conditions. Earlier work has demonstrated that low nutrition scores can independently predict mortality in the LVAD population. This study explored changes in MNA scores and other clinical markers following LVAD. METHODS: This retrospective study included 74 patients implanted with LVADs between 2012 and 2017. MNA or MNA-SF along with other clinical data and nutrition indices were assessed during the preoperative workup and reassessed on average 423.9 days post LVAD. Paired-samples t-tests were used to evaluate any changes. RESULTS: Despite an average body mass index of 30.8, 28.3% of patients were classified by MNA as malnourished, and 58.5% were considered at risk prior to LVAD implantation. Post LVAD implantation, MNA scores improved from an average of 19.2-23.0 (P < 0.001), with now only 3.8% classified as malnourished and 45.3% classified as at risk. MNA-SF and prognostic nutritional index also improved significantly. CONCLUSIONS: This study indicates that LVAD implantation is associated with a long-term improvement in nutrition status when compared with the preoperative heart failure state.

Effects of prostaglandin E2 on gap junction protein alpha 1 in the rat epididymis.

Gap junctions are responsible for intercellular communication. In the adult mammalian epididymis, gap junction protein alpha 1 (GJA1) is localized between basal and either principal or clear cells. GJA1 levels and localization change during the differentiation of basal cells. The present objective was to determine the role of basal cells and prostaglandin E2 (PGE2) on GJA1 in the rat epididymis. Prior to basal cell differentiation, GJA1 is colocalized with TJP1 at the apical lateral margins between adjacent epithelial cells. When basal cells are present, GJA1 becomes associated between basal and principal cells, where it is primarily immunolocalized until adulthood. Basal cells express TP63, differentiate from epithelial cells, and produce prostaglandin-endoperoxide synthase 1 by 21 days of age. Prior to day 21, GJA1and TP63 are not strongly associated at the apical region. However, by day 28, TP63-positive basal cells migrate to the base of the epithelium, and also express GJA1. To assess effects of PGE2 on GJA1, rat caput epididymal (RCE) cells were exposed to PGE2 (50 µM) for 3 h. PGE2 increased levels of Gja1 mRNA in RCE cells, while levels of Gjb1, Gjb2, Gjb4, and GjB5 were unaltered. Furthermore, PGE2 increased protein levels of GJA1, phospho-GJA1, phospho-AKT, CTNNB1, and phospho-CTNNB1. Total AKT and the tight junction protein claudin1 were also not altered by PGE2. Data suggest that development of the epididymal epithelium and differentiation of epididymal basal cells regulate the targeting of GJA1, and that this appears to be mediated by PGE2.

Cellular junctions in the epididymis, a critical parameter for understanding male reproductive toxicology.

Epididymal sperm maturation is a critical aspect of male reproduction in which sperm acquire motility and the ability to fertilize an ovum. Sperm maturation is dependent on the creation of a specific environment that changes along the epididymis and which enables the maturation process. The blood-epididymis barrier creates a unique luminal micro-environment, different from blood, by limiting paracellular transport and forcing receptor-mediated transport of macromolecules across the epididymal epithelium. Direct cellular communication between cells allows coordinated function of the epithelium. A limited number of studies have directly examined the effects of toxicants on junctional proteins and barrier function in the epididymis. Effects on the integrity of the blood-epididymis barrier have resulted in decreased fertility and, in some cases, the development of sperm granulomas. Studies have shown that in addition to tight junctions, proteins implicated in the maintenance of adherens junctions and gap junctions alter epididymal functions. This review will provide an overview of the types and roles of cellular junctions in the epididymis, and how these are targeted by different toxicants.

Fathers' Stress in the Neonatal Intensive Care Unit: A Systematic Review.

BACKGROUND: Admission to the neonatal intensive care unit (NICU) is stressful for parents. Nurses often focus on maternal well-being and fail to acknowledge the stress of fathers. Research on fathers' psychological stress is limited. PURPOSE: A systematic review of the literature was completed to examine the extent of psychological stress and types of stressors in fathers with infants admitted to the NICU. METHODS/SEARCH STRATEGY: A search of Ovid MEDLINE, Cochrane Library, PsycINFO, CINAHL, and EMBASE was conducted to identify descriptive and observational studies reporting father-specific stress in the NICU. Studies using observational and descriptive designs, published in English, and reporting father-specific stress outcomes during a NICU admission were eligible for inclusion. Strengthening the Reporting of Observational Studies in Epidemiology guidelines were used for quality assessment. RESULTS: Fifteen studies met inclusion criteria. Fathers find the NICU environment stressful and are more stressed than fathers of full-term, healthy infants. Parental role alteration, infant appearance, NICU environment, and staff communication are stressors. IMPLICATIONS FOR PRACTICE/RESEARCH: By recognizing the extent and types of psychological stress in fathers, nurses can provide better support for fathers in their new role. Younger fathers and those with very low birth-weight premature infants may need additional support and resources. Future research on fathers' stress should include larger sample sizes, diverse populations, and tool development and evaluation.

Quality of Life for Parents of Children With Congenital Heart Defect: A Systematic Review.

BACKGROUND: As survival rates for infants born with severe forms of cardiac defects (congenital heart defect [CHD]) improve, attention is directed to evaluating factors that affect the child's short- and long-term outcomes including parental quality of life (QOL). PURPOSE: The purpose of this review was to identify how parental QOL is affected when having a child with a CHD. Factors that influence parental QOL when having a child with a CHD will also be described. METHODS: A systematic search of CINAHL, EMBASE, PsycINFO, and PubMed databases was performed. Thirty-three quantitative cross-sectional or cohort studies were selected for inclusion and analyzed for quality reporting using Strengthening the Reporting of Observational Studies in Epidemiology guidelines. RESULTS: Heart defect severity, age of child, perceived support, and availability of economic resources were identified as factors affecting parental QOL. Parent gender was related to QOL and family functioning factors. Paternal outcomes were reported in 23 of the 33 studies (70%), with an average father participation rate of 40%. CONCLUSIONS: Having a child with CHD negatively affects parental QOL. Future research should include targeting fathers to improve understanding of their unique perceptions and needs. Longitudinal studies should also describe correlations of parental QOL with their child's developmental outcomes. Efficacy studies testing supportive interventions on outcomes such as improved adjustment and QOL are needed.

Betamethasone causes intergenerational reproductive impairment in male rats.

Prenatal betamethasone (BM) exposure in rats negatively impacts sperm quality and male fertility. Studies have shown that BM can cause multi-generational effects on the pituitary-adrenal-axis of rats. The objective of this study was to assess the reproductive development and fertility of male rats (F2) whose fathers (F1) were exposed to BM (0.1mg/kg) on gestational days 12, 13, 18 and 19. In F2 rats, there was a significant reduction in body weights of the BM-treated group at PND 1 as well as delayed onset of puberty, and decreases in FSH levels, Leydig cell volume, sperm number and motility, seminal vesicle contractility and ejaculated volume. Furthermore, increased serum LH levels, sperm DNA damage and abnormal morphology were observed, resulting in reduced fertility. In conclusion, prenatal BM-treatment leads to intergenerational long-term reproductive impairment in male rats, raising concern regarding the widespread use of BM in preterm births.

Alterations in male rats following in utero exposure to betamethasone suggests changes in reproductive programming.

Antenatal betamethasone is used for accelerating fetal lung maturation for women at risk of preterm birth. Altered sperm parameters were reported in adult rats after intrauterine exposure to betamethasone. In this study, male rat offspring were assessed for reproductive development after dam exposure to betamethasone (0.1mg/kg) or vehicle on Days 12, 13, 18 and 19 of pregnancy. The treatment resulted in reduction in the offspring body weight, delay in preputial separation, decreased seminal vesicle weight, testosterone levels and fertility, and increased testicular weight. In the testis, morphologically abnormal seminiferous tubules were observed, characterized by an irregular cell distribution with Sertoli cell that were displaced towards the tubular lumen. These cells expressed both Connexin 43 (Cx43) and Proliferative Nuclear Cell Antigen (PCNA). In conclusion, intrauterine betamethasone treatment appears to promote reproductive programming and impairment of rat sexual development and fertility due to, at least in part, unusual testicular disorders.

Implications of caveolae in testicular and epididymal myoid cells to sperm motility.

Seminiferous tubules of the testis and epididymal tubules in adult rodents are enveloped by contractile myoid cells, which move sperm and fluids along the male reproductive tract. Myoid cells in the testis influence Sertoli cells by paracrine signaling, but their role in the epididymis is unknown. Electron microscopy revealed that elongated myoid cells formed several concentric layers arranged in a loose configuration. The edges of some myoid cells in a given layer closely approximated one another, and extended small foot-like processes to cells of overlying layers. Gap junction proteins, connexins 32 and 43, were detected within the myoid cell layers by immunohistochemistry. These myoid cells also had caveolae that contained caveolin-1 and cavin-1 (also known as PTRF). The number of caveolae per unit area of plasma membrane was significantly reduced in caveolin-1-deficient mice (Cav1(-/-) ). Morphometric analyses of Cav1-null testes revealed an enlargement in whole-tubule and epithelial profile areas, whereas these parameters were slightly reduced in the epididymis. Although sperm are non-motile as they pass through the proximal epididymis, statistical analyses of cauda epididymidis sperm concentrations revealed no significant differences between wild-type and Cav1(-/-) mice. Motility analyses, however, indicated that sperm velocity parameters were reduced while beat cross frequency was higher in gametes of Cav1(-/-) mice. Thus while caveolae and their associated proteins are not necessary for myoid cell contractility, they appear to be crucial for signaling with the epididymal epithelium to regulate the proper acquisition of sperm motility. Mol. Reprod. Dev. 83: 526-540, 2016. © 2016 Wiley Periodicals, Inc.

Nutrition Assessment With Indirect Calorimetry in Patients Evaluated for Left Ventricular Assist Device Implantation.

BACKGROUND: Malnutrition is known to negatively impact the clinical course of advanced heart failure and is associated with increased mortality following left ventricular assist device (LVAD) implantation. Appropriate assessment of nutrition requirements in these patients is critical in their clinical care, yet there has been little discussion on how to best determine resting energy expenditure (REE) in the hospital setting. We investigated the use of indirect calorimetry in a group of patients with advanced heart failure. MATERIALS AND METHODS: Results from preoperative indirect calorimetry testing in 98 patients undergoing evaluation for LVAD candidacy were collected. REE was compared with 10 predictive equations that estimated caloric need based on a range of patient-specific demographic and clinical variables. RESULTS: This study enrolled 22 female and 76 male patients with a mean age of 59.4 ± 12.5 years, body mass index of 29.6 ± 6.0 kg/m(2), and ejection fraction of 19.4 ± 6.6%. The average REE by indirect calorimetry in this group was 1610.0 ± 612.7 kcal/d. All predictive equations significantly overestimated REE. However, those equations intended for use in the critically ill demonstrated the greatest accuracy, with the Brandi equation achieving both the highest correlation (r = 0.605, P < .001) and the lowest standard error of the estimate (504.8 kcal/d). CONCLUSIONS: Indirect calorimetry may be reliably and safely used to determine caloric requirements in patients with advanced heart failure. The use of predictive equations based on demographic and clinical parameters appears to generate inaccurate estimations of REE in these patients. However, equations designed for use in critically ill patients better estimate nutrition requirements than those designed for healthy individuals.

Claudin-10 is required for relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

Species-specific symmetry-breaking events at the left-right organizer (LRO) drive an evolutionarily-conserved cascade of gene expression in the lateral plate mesoderm that is required for the asymmetric positioning of organs within the body cavity. The mechanisms underlying the transfer of the left and right laterality information from the LRO to the lateral plate mesoderm are poorly understood. Here, we investigate the role of Claudin-10, a tight junction protein, in facilitating the transfer of left-right identity from the LRO to the lateral plate mesoderm. Claudin-10 is asymmetrically expressed on the right side of the chick LRO, Hensen's node. Gain- and loss-of-function studies demonstrated that right-sided expression of Claudin-10 is essential for normal rightward heart tube looping, the first morphological asymmetry during organogenesis. Manipulation of Claudin-10 expression did not perturb asymmetric gene expression at Hensen's node, but did disrupt asymmetric gene expression in the lateral plate mesoderm. Bilateral expression of Claudin-10 at Hensen's node prevented expression of Nodal, Lefty-2 and Pitx2c in the left lateral plate mesoderm, while morpholino knockdown of Claudin-10 inhibited expression of Snail1 in the right lateral plate mesoderm. We also determined that amino acids that are predicted to affect ion selectivity and protein interactions that bridge Claudin-10 to the actin cytoskeleton were essential for its left-right patterning function. Collectively, our data demonstrate a novel role for Claudin-10 during the transmission of laterality information from Hensen's node to both the left and right sides of the embryo and demonstrate that tight junctions have a critical role during the relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

Short-form nutrition assessment in patients with advanced heart failure evaluated for ventricular assist device placement or cardiac transplantation.

BACKGROUND: It has been shown that malnutrition affects clinical outcomes in patients with advanced heart failure and that nutrition status, as determined by the Mini Nutritional Assessment (MNA), can be used as an independent predictor of mortality. The aim of this study was to evaluate the prognostic utility of the short-form MNA (MNA-SF) as a surrogate to the MNA in patients with advanced heart failure. METHODS: Data retrospectively gathered from nutrition assessments of 162 patients were analyzed. RESULTS: As defined by the MNA, the cohort included 40 (24.7%) patients classified as malnourished, 106 (65.4%) classified as at risk, and 16 (9.9%) classified as well nourished. The mortality for the groups was 37.3%, 47.4%, and 40.5%, respectively. A linear regression showed strong correlation between the MNA and MNA-SF (r = 0.778, P < .0001). A significant difference was observed in survival between the undernourished state (at risk + malnourished) and the well-nourished state, as determined by the MNA-SF (P < .001). CONCLUSIONS: The MNA-SF is a rapid nutrition assessment that correlates strongly with the full-form MNA and is an independent predictor of mortality.

The blood-epididymis barrier and inflammation.

The blood-epididymis barrier (BEB) is a critical structure for male fertility. It enables the development of a specific luminal environment that allows spermatozoa to acquire both the ability to swim and fertilize an ovum. The presence of tight junctions and specific cellular transporters can regulate the composition of the epididymal lumen to favor proper sperm maturation. The BEB is also at the interface between the immune system and sperm. Not only does the BEB protect maturing spermatozoa from the immune system, it is also influenced by cytokines released during inflammation, which can result in the loss of barrier function. Such a loss is associated with an immune response, decreased sperm functions, and appears to be a contributing factor to post-testicular male infertility. Alterations in the BEB may be responsible for the formation of inflammatory conditions such as sperm granulomas. The present review summarizes current knowledge on the morphological, physiological and pathological components associated with the BEB, the role of immune function on the regulation of the BEB, and how disturbance of these factors can result in inflammatory lesions of the epididymis.

Nutrition assessment in advanced heart failure patients evaluated for ventricular assist devices or cardiac transplantation.

BACKGROUND: Malnutrition has been shown to affect clinical outcomes in patients with heart failure. The aim of this study was to analyze the incidence of malnutrition and to assess its prognostic significance in patients with advanced heart failure (AHF) (being evaluated for left ventricular assist device [LVAD] or cardiac transplant) based on nutrition status as assessed by the Mini Nutritional Assessment (MNA). METHODS: A retrospective analysis was conducted on 154 patients. During evaluation, a complete nutrition assessment was performed, and diagnosis of malnutrition and risk of malnutrition was done with the MNA. Its possible independent association with mortality was assessed. RESULTS: The mean (SD) age of the patients was 59.3 (14.1) years, with 76% men. Twenty-two percent were classified as malnourished, 68% at risk of malnutrition, and 10% well nourished. The mortality in the 3 groups was 26.5%, 42.0%, and 6.7%, respectively (P = .02). In the multivariate logistic regression analysis, the undernutrition state (malnourished + at risk) was an independent predictor of mortality (odds ratio, 7.9; confidence interval, 1.01-62.30; P = .04). CONCLUSIONS: The state of undernutrition is an independent predictor of mortality in patients with AHF. Early recognition of undernutrition through use of the MNA may affect the long-term prognosis of these patients by enabling early intervention.

Everolimus and intensive behavioral therapy in an adolescent with tuberous sclerosis complex and severe behavior.

BACKGROUND: Self-injury and aggression have been reported in individuals with TSC (tuberous sclerosis complex), yet few data exist about treatment. Everolimus, an mTOR inhibitor, has been FDA-approved for subependymal giant cell astrocytomas (SEGAs) and renal angiomyolipomas in TSC. However, clinical use of everolimus with direct, real-time observations of self-injury and aggression in an individual with TSC has not been reported. METHODS: During an inpatient admission to a neurobehavioral unit, real-time measurements of behaviors and seizures were recorded. An interdisciplinary team used these data to make treatment decisions and applied behavioral and pharmacological treatments, one at a time, in order to evaluate their effects. RESULTS: Aggression and self-injury improved with applied behavioral analysis (ABA), lithium, and asenapine. Improvements in SEGA size, facial angiofibromas, seizures, and the most stable low rates of self-injury were observed during the interval of treatment with everolimus. CONCLUSION: Mechanism-based treatments in the setting of an evidence-based behavioral and psychopharmacological intervention program may be a model with utility for characterization and treatment of individuals with severe behavior and TSC.