Jennifer L. Kirby

Ischemia-Reperfusion of the Murine Testis Stimulates the Expression of Proinflammatory Cytokines and Activation of c-jun N-Terminal Kinase in a Pathway to E-Selectin Expression

Abstract Ischemia-reperfusion (IR) of the testis results in germ cell-specific apoptosis and can lead to aspermatogenesis. Germ cell-specific apoptosis after IR of the testis has been shown to be correlated with and dependent on neutrophil recruitment to the testis after IR. Studies that used E-selectin-deficient mice have demonstrated that E-selectin expression is critical for neutrophil recruitment to subtunical venules in the testis after IR and for the resultant germ cell-specific apoptosis. The present study investigates the in vivo signaling pathway that exists after IR that leads to neutrophil recruitment in the murine testis. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. Results demonstrate that the proinflammatory cytokines, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β), are stimulated after IR as is the phosphorylation of c-jun N-terminal kinase (JNK). The downstream transcription factors of JNK, ATF-2 and c-jun are also phosphorylated at specific times after IR of the testis. Activation of the JNK stress-related kinase pathway is correlated with an increase in E-selectin expression and neutrophil recruitment to the testis after IR. Intratesticular injection of IL-1β also caused JNK phosphorylation and neutrophil recruitment to the testis. These results suggest that testicular IR injury stimulates IL-1β expression, which leads to activation of the JNK signaling pathway and ultimately E-selectin expression and neutrophil recruitment to the testis. This provides the first evidence of a cytokine/stress-related kinase signaling pathway to E-selectin expression in vivo.

The Development of the Epididymis

The adult epididymis is a highly convoluted tubule that performs a variety of functions including sperm protection, maturation, concentration and storage. These functions can only be accomplished following a series of morphological changes that occur during development. These changes result in the development of a single tubule that differs in several regions with respect to morphology, function and gene expression. Therefore, it is not surprising that the development of the epididymis is a highly regulated event and is not yet completely understood. This chapter will attempt to summarize the events that take place leading to the development and differentiation of the adult epididymis. Major key studies will be summarized and important questions that remain unanswered will be addressed.

Characterization of Fibroblast Growth Factor Receptors Expressed in Principal Cells in the Initial Segment of the Rat Epididymis

Abstract Studies from our laboratory support a model in which growth factors produced in the testis reach the epididymis via the luminal system and play an important role in maintaining the function of epithelial cells, particularly in the initial segment. Previous work showed that γ-glutamyl transpeptidase (GGT) mRNA IV, which is highly expressed in the rat initial segment, may be under the control of luminal fibroblast growth factor 2 (FGF-2) from the testis. The current studies were undertaken to identify which fibroblast growth factor receptors (FGFRs) are present in the principal cells of the rat initial segment and to identify other potential ligands for these receptors in rat rete testis fluid (RTF). Immunoblot analysis revealed that FGFRs 1–4 were present, and reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that both the IIIb and IIIc splice variants of FGFRs 1–3 were expressed. However, RT-PCR using RNA isolated from principal cells collected by laser capture microdissection revealed only FGFR-1 IIIc. Additional PCR analysis established that both the α and β forms of FGFR-1 IIIc were expressed in principal cells. Both FGF-4 and FGF-8 were present in rat RTF, as determined by immunoblotting. Thus, FGF-2, -4, and -8, found in RTF, may act upon FGFR-1 IIIc in the principal cells of the initial segment to regulate GGT mRNA IV expression.

Hypoxia-Inducible Factor-1α Is Constitutively Expressed in Murine Leydig Cells and Regulates 3β-Hydroxysteroid Dehydrogenase Type 1 Promoter Activity

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that plays an essential role in oxygen homeostasis. HIF-1alpha is constitutively made in cells; however, it is ubiquitinated and degraded under normoxic conditions. Hypoxia prevents the ubiquitination of HIF-1alpha, resulting in stabilization of the protein and activation of target genes. Because of its vascular arrangement and the high metabolic demand of spermatogenesis, the testis has been described previously as functioning on the brink of hypoxia; thus, we have hypothesized that HIF-1alpha is constitutively expressed and stabilized in the testis, where it could play a role in testicular homeostasis. Western blot analysis using nuclear proteins from liver, kidney, and testis revealed the presence of HIF-1alpha only in the testis. Immunohistochemistry confirmed this result and revealed that HIF-1alpha was specifically located in interstitial Leydig cells. Electromobility shift assays employing nuclear extracts from the TM3 Leydig cell line revealed that these cells express HIF-1alpha that is capable of binding DNA under normoxic conditions. Furthermore, we found that protein levels can be increased further when the TM3 cells are cultured under hypoxic conditions. Finally, transient transfections of TM3 Leydig cells revealed that the promoter of the mouse 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1) gene, which encodes a key enzyme in testosterone production, is a potential target of HIF-1alpha. In conclusion, HIF-1alpha is constitutively present in the Leydig cells of the murine testis, where it potentially regulates Hsd3b1 transcription, and thus male reproductive function.

Inhibitor of Differentiation-3 Mediates High Fat Diet-Induced Visceral Fat Expansion

Objective—Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high-fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in HFD-induced angiogenesis and visceral adipose expansion is unknown. Methods and Results—Id3−/− mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to wild type littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to wild type controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction and Id3−/− mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to wild type. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity. Conclusion—Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

Putative Regulation of Expression of Members of the Ets Variant 4 Transcription Factor Family and Their Downstream Targets in the Rat Epididymis

Abstract Several genes expressed in the initial segment of the epididymis depend on factors from the testis that reach the epididymis via the luminal system. These include gamma-glutamyl transpeptidase mRNA IV (Ggt_pr4), steroid 5 alpha reductase (Srd5a1), glutathione peroxidase 5 (Gpx5), and cystatin-related epididymal spermatogenic (Cst8) genes. Promoter analyses indicated that these genes contain several ETS DNA-binding sites. Members of the polyomavirus enhancer activator 3 (ETV4) family bind to ETS sites on the promoter of target genes to regulate transcription. In this study, the role of ETV4 family members (ETV4, ETV5, ETV1) in the transcription of initial segment specific genes was evaluated. All three ETV4 family mRNAs are expressed in the principal cells of the initial segment and depend upon the presence of testicular luminal fluid factors. ETV4 protein was localized to principal cell nuclei and displayed the highest expression in the most proximal region of the initial segment. In addition, ETV4 protein levels were diminished after loss of testicular luminal fluid factors. A dominant-negative construct of ETV5 was in vivo electroporated into the initial segment to determine if ETV4 family members can regulate the transcription of testicular luminal fluid factor-regulated genes. Quantitative PCR indicated that 1 day postelectroporation, all three ETV4 family member mRNAs were significantly decreased. In addition, Ggt_pr4, Srd5a1, and Gpx5 mRNA levels were also significantly decreased. The data suggest that ETV4 family members regulate their own expression, and that they regulate transcription of a subset of genes that are dependent upon testicular luminal fluid factors.

Characterization of Epididymal Epithelial Cell-Specific Gene Promoters by In Vivo Electroporation

Abstract The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and γ-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5′ site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between −530 and −681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.

Bariatric surgery insurance requirements independently predict surgery dropout

BACKGROUND Many insurance companies have considerable prebariatric surgery requirements despite a lack of evidence for improved clinical outcomes. The hypothesis of this study is that insurance-specific requirements will be associated with a decreased progression to surgery and increased delay in time to surgery. METHODS Retrospective data collection was performed for patients undergoing bariatric surgery evaluation from 2010-2015. Patients who underwent surgery (SGY; n = 827; mean body mass index [BMI] 49.1) were compared with those who did not (no-SGY; n = 648; mean BMI: 49.4). Univariate and multivariate analysis were performed to identify specific co-morbidity and insurance specific predictors of surgical dropout and time to surgery. RESULTS A total of 1475 patients using 12 major insurance payors were included. Univariate analysis found insurance requirements associated with surgical drop out included longer median diet duration (no-SGY = 6 mo; SGY = 3 mo; P<.001); primary care physician letter of necessity (P<.0001); laboratory testing (P = .019); and evaluation by cardiology (P<.001), pulmonology (P<.0001), or psychiatry (P = .0003). Using logistic regression to control for co-morbidities, longer diet requirement (odds ratio [OR] .88, P<.0001), primary care physician letter (OR .33, P<.0001), cardiology evaluation (OR .22, P = .038), and advanced laboratory testing (OR 5.75, P = .019) independently predicted surgery dropout. Additionally, surgical patients had an average interval between initial visit and surgery of 5.8±4.6 months with significant weight gain (2.1 kg, P<.0001). CONCLUSION Many prebariatric surgery insurance requirements were associated with lack of patient progression to surgery in this study. In addition, delays in surgery were associated with preoperative weight gain. Although prospective and multicenter studies are needed, these findings have major policy implications suggesting insurance requirements may need to be reconsidered to improve medical care.

Clinical significance of failure to lose weight 10 years after roux-en-y gastric bypass

BACKGROUND Although Roux-en-Y gastric bypass (RYGB) induces short-term weight loss and co-morbidity amelioration, long-term data suggest that a subset of patients return to their preoperative body mass index (BMI). OBJECTIVES To identify the clinical implications of 10-year weight loss failure after RYGB. SETTING An academic teaching hospital. METHODS Adults undergoing RYGB (1985-2004) were included in this study (n = 1087). Absolute weight loss failure was defined as ≤0% reduction in excess BMI 10 years after surgery. Univariate analyses compared co-morbidity rates and resolution by weight loss classification. Multivariable regression modeling analyzed preoperative predictors of 10-year percent reduction in excess BMI and weight loss failure. RESULTS Complete follow-up was available for 617 (57%) patients with a 10-year median percent reduction in excess BMI of 57.1%; 10.2% of patients had weight loss failure. Prevalence of all co-morbidities decreased, even in patients with weight loss failure (all P<.05). Compared with patients with successful weight loss, patients with weight loss failure had similar rates of resolution of pre-existing co-morbidities, except for reduced resolution of apnea and cardiac co-morbidities (both P<.05). Risk factors for weight loss failure included lower BMI, nongovernmental insurance, longer travel time to hospital, and year of surgery. Nongovernmental insurance (odds ratio 2.03, P = .036) conferred the highest adjusted odds of weight loss failure. CONCLUSIONS The vast majority of patients experience dramatic health improvement 10 years after RYGB, even though some patients fail to maintain their weight loss. Renewed focus should be placed on prevention and treatment of chronic disease, with further investigation of weight loss independent mechanisms of health improvement.

Bariatric Surgery Resistance: Using Preoperative Lifestyle Medicine and/or Pharmacology for Metabolic Responsiveness.

Bariatric surgery is an effective and durable treatment for individuals with obesity and its associated comorbidities. However, not all patients meet weight loss and/or cardiometabolic goals following bariatric surgery, suggesting that some people are bariatric surgery resistant. The reason for this resistance is unclear, but potential factors, such as adiposity-derived inflammation, insulin resistance, hyperglycemia, and aerobic fitness prior to surgery, have been related to blunted surgery responsiveness. Exercise, diet, and/or pharmacology are effective at reducing inflammation and improving insulin action as well as physical function. Herein, we present data that supports the novel hypothesis that intervening prior to surgery can enhance disease resolution in people who are resistant to bariatric surgery.