Proteomics identifies differences in fibrotic potential of extracellular vesicles from human tendon and muscle fibroblasts.

BACKGROUND: Fibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis. METHODS: Here, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs. RESULTS: Mass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFß1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix. CONCLUSIONS: Our data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans. Video abstract.

External validity of a model to predict postoperative atrial fibrillation after thoracic surgery.

OBJECTIVES: A prediction model developed by Passman et al. stratifies patients' risk of postoperative atrial fibrillation (POAF) after major non-cardiac thoracic surgery using 3 simple factors (sex, age and preoperative resting heart rate). The model has neither undergone external validation nor proven to be relevant in current thoracic surgery practice. METHODS: A retrospective single-centre analysis of all patients who underwent major non-cardiac thoracic surgery (2008-2017) with prospective documentation of incidence and severity of POAF was used for external validation of Passman's derivation sample (published in 2005 with 856 patients). The model calibration was assessed by evaluating the incidence of POAF and patients' risk scores (0-6). RESULTS: A total of 2054 patients were included. Among them, POAF occurred in 164 (7.9%), compared to 147 (17.2%) in Passman's study. Differences in our sample compared to Passman's sample included mean heart rate (75.7 vs 73.7 bpm, P < 0.001), proportion of patients with hypertension (46.1 vs 29.4%, P < 0.001), proportion of extensive lung resections, particularly pneumonectomy (6.1 vs 21%, P < 0.001) and proportion of minimally invasive surgeries (56.6% vs 0%). The model demonstrated a positive correlation between risk scores and POAF incidence (risk score 1.2% vs 6.16%). CONCLUSIONS: The POAF model demonstrated good calibration in our population, despite a lower overall incidence of POAF compared to the derivation study. POAF rates were higher among patients with a higher risk score and undergoing procedures with greater intrathoracic dissection. This tool may be useful in identifying patients who are at risk of POAF when undergoing major thoracic surgery and may, therefore, benefit from targeted prophylactic therapy.

Long-Term Results of Surgically Treated Radial Polydactyly - An Outcome Correlation Study.

Background: Thumb polydactyly is one of the commonest congenital hand differences. Traditional surgeon-based outcome scores capture outcomes mainly on bodily structure and function. Outcomes on the long-term well-being of the patients in the domains of activity and participation are not fully studied. Methods: Forty-eight thumbs in forty-five Chinese patients with radial polydactyly underwent surgical treatment at or before 3 years old were recruited. Mean follow-up was 11.6 years. Surgical outcomes were collected and compared to the normal opposite thumb. The results were compiled into the Japanese Society for Surgery of the Hand (JSSH) score, Cheng score and Tada score. Patients' activity involving hands were assessed by both objective tools and patient-reported outcome measure while their health-related quality of life (HRQoL) was assessed by Patient- and Parent-reported Pediatric Quality of Life Inventory (PedsQL). Correlations between outcomes were analysed. Results: Overall, both parents and patients themselves reported good quality of life with mean score of 86.6% and 92.1% respectively in PedsQL. The combined surgical scores ranged from 52% good or excellent results using JSSH score to 100% good result using Cheng score. None of the outcomes on bodily structure and function showed positive correlation with patient's well-being. Negative correlation was noted in total passive range of movement, active movement and Cheng score. All patients reported no activity restriction. Writing test did not show significant slowing. The operated hands had significantly poorer fine motor dexterity than normal. No significant correlation is noted between activity outcomes and PedsQL. Conclusions: Outcomes on bodily structure, function and activity showed little correlation with patients' well-being after thumb polydactyly correction. It should be careful in using or analysing patient/parent-reported outcome measures on HRQoL as outcome assessment of surgical treatment of radial polydactyly.

Forecasting pulmonary air leak duration following lung surgery using transpleural airflow data from a digital pleural drainage device.

BACKGROUND: Prolonged air leak (PAL) is often the limiting factor for hospital discharge after lung surgery. Our goal was to develop a statistical model that reliably predicts pulmonary air leak resolution by applying statistical time series modeling and forecasting techniques to digital drainage data. METHODS: Autoregressive Integrated Moving Average (ARIMA) modeling was used to forecast air leak flow from transplural air flow data. The results from ARIMA were retrospectively internally validated with a group of 100 patients who underwent lung resection between December 2012 and March 2017, for whom digital pleural drainage data was available for analysis and a persistent air leak was the limiting factor for chest tube removal. RESULTS: The ARIMA model correctly identified 82% (82/100) of patients as to whether or not the last chest tube removal was appropriate. The performance characteristics of the model in properly identifying patients whose air leak would resolve and who would therefore be candidates for safe chest tube removal were: sensitivity 80% (95% CI, 69-88%), specificity 88% (95% CI, 68-97%), positive predictive value 95% (95% CI, 86-99%), and negative predictive value 59% (95% CI, 42-79%). The false positive and false negative rate was 12% (95% CI, 12-31%) and 20% (95% CI, 12-31%). CONCLUSIONS: We were able to validate a statistical model that that reliably predicted resolution of pulmonary air leak resolution over a 24-hour period. This information may improve the care of patients with chest tube by optimizing duration of pleural drainage.

A review and analysis of strategies for prediction, prevention and management of post-operative atrial fibrillation after non-cardiac thoracic surgery.

Atrial fibrillation (AF) is the most common sustained arrhythmia after non-cardiac thoracic surgery and is associated with a significant increase in perioperative morbidity, intensive care unit (ICU) admission, and mortality. Practical guidance is needed to assist clinicians in managing this critical issue and direct further research. Here we aim to provide a synoptic review and analysis of the literature to distil practical recommendations for prediction, prevention and management of post-operative atrial fibrillation (POAF) suitable for clinical application and further evaluation. To predict POAF, risk factors including age, gender, elevated pre-operative heart rate and extent of surgical resection have been reproducibly identified and integrated into scoring systems. To prevent POAF, prophylactic therapy with beta-blockers, amiodarone, or magnesium have demonstrated to be effective, but need further trials in high-risk populations. To manage unstable POAF that precipitates hypotension and hypoperfusion, although rare, requires immediate electrocardioversion to restore cardiac output and adequate oxygen delivery. For hemodynamically stable patients, rate control and prevention of adverse events are the objectives. We propose an individualized approach aimed at rate control using initial incremental low dose beta-blocker or calcium channel blocker (CCB) therapy with close monitoring of a patient's response, and continuation of the drug that they respond to, along with simultaneous identification and reduction of triggers of AF, in order for spontaneous return to sinus rhythm. For patients who persistently fail to respond to rate control therapy, rhythm control may be considered using an agent selected based on the patient's comorbidities and the medications' side effect profile. While controversial and requiring further study, anticoagulation therapy is recommended in patients with risk factors for thromboembolic events after 48 hours of persistent AF. We recommend continuous prospective monitoring of incidence and severity of POAF to track the impact of protocols to predict, prevent and manage POAF.

The state of uniportal video-assisted thoracoscopic surgery in North America: a survey of thoracic surgeons.

BACKGROUND: In recent years, there has been an exponential growth in the research and development of uniportal video-assisted thoracoscopic surgery (VATS). In the context of the 2017 Annual Asian Single-Port VATS Symposium held in Shanghai, China, we sought to describe current the state of uniportal VATS in North America and explore factors that could influence future adoption. METHODS: In March 2017, a five-question survey was distributed to North American Thoracic Surgeons in order to obtain their opinion regarding the uniportal VATS approach to pulmonary resection. Responses were summarized and statistical comparisons of categorical variables were performed using Fisher's exact test. RESULTS: The estimated response rate to the survey was 16.5% (99/600). The majority of respondents were experienced surgeons with 41.4% (41/99) having been in practice 11-20 years. The majority (70%; 69/99) of surgeons had never performed a uniportal VATS procedure. When surgeons were asked to state what could potentially convince them to adopt uniportal VATS, scientific evidence of superiority (86%; 85/99) and/or of superior ergonomics (39%; 39/99), attendance to focused conferences with a practical simulation component (38%; 38/99), and the availability of surgical proctorship (9%; 9/99) were the most commonly selected responses. CONCLUSIONS: This survey is the first of its kind to provide a glimpse into the status of the uniportal VATS in North America. The responses suggest that there are few early adopters of this approach as compared to other parts of the world. The lack of perceived advantages to uniportal VATS and the need for more comparative evidence to other established approaches appear to be major obstacles to more widespread adoption in North America.

An ex vivo porcine skin model to evaluate pressure-reducing devices of different mechanical properties used for pressure ulcer prevention.

Pressure ulcers are complex wounds caused by pressure- and shear-induced trauma to skin and underlying tissues. Pressure-reducing devices, such as dressings, have been shown to successfully reduce pressure ulcer incidence, when used in adjunct to pressure ulcer preventative care. While pressure-reducing devices are available in a range of materials, with differing mechanical properties, understanding of how a material's mechanical properties will influence clinical efficacy remains limited. The aim of this study was to establish a standardized ex vivo model to allow comparison of the cell protection potential of two gel-like pressure-reducing devices with differing mechanical properties (elastic moduli of 77 vs. 35 kPa). The devices also displayed differing energy dissipation under compressive loading, and resisted strain differently under constant load in compressive creep tests. To evaluate biological efficacy we employed a new ex vivo porcine skin model, with a confirmed elastic moduli closely matching that of human skin (113 vs. 119 kPa, respectively). Static loads up to 20 kPa were applied to porcine skin ex vivo with subsequent evaluation of pressure-induced cell death and cytokine release. Pressure application alone increased the percentage of epidermal apoptotic cells from less than 2% to over 40%, and increased cellular secretion of the pro-inflammatory cytokine TNF-alpha. Co-application of a pressure-reducing device significantly reduced both cellular apoptosis and cytokine production, protecting against cellular damage. These data reveal new insight into the relationship between mechanical properties of pressure-reducing devices and their biological effects. After appropriate validation of these results in clinical pressure ulcer prevention with all tissue layers present between the bony prominence and external surface, this ex vivo porcine skin model could be widely employed to optimize design and evaluation of devices aimed at reducing pressure-induced skin damage.

Comparison of gene expression of the oncogenic Wnt/ß-catenin signaling pathway components in the mouse and human epididymis.

ß-catenin is an integral part of the Wnt signaling pathway and has been linked to tumorigenesis and multiple developmental processes. The high ß-catenin expression with low tumor incidence in the human epididymis is thus intriguing. In the present study, the ß-catenin gene and protein was found to be highly expressed in the murine caput epididymidis, and the protein mainly localized along the lateral plasma membranes of adjacent epithelial cells throughout both human and mouse epididymides. Furthermore, the adult mouse epididymis was found to express almost all the Wnt/ß-catenin signaling pathway genes that were determined previously by our group in the human organ. Despite the differences in epididymal structure, the similar location of ß-catenin and the high concordance of this pathway's components' gene expression in both the adult human and mouse epididymides make the mouse a suitable animal model for studying the anti-tumor mechanism of the epididymis. In addition, both the mRNA and protein expression of ß-catenin shared a similar spatial expression as the mRNA of Ros1, a proto-oncogene and a key developmental regulator of the initial segment of the mouse epididymis. The observations on the parallel temporal expression of ß-catenin and Ros1 during postnatal development raise the possibility that the canonical Wnt signaling pathway has an additional role in the postnatal development of mouse epididymis.

Arhgap28 is a RhoGAP that inactivates RhoA and downregulates stress fibers.

The small GTPase RhoA is a major regulator of actin reorganization during the formation of stress fibers; thus identifying molecules that regulate Rho activity is necessary for a complete understanding of the mechanisms that determine cell contractility. Here, we have identified Arhgap28 as a Rho GTPase activating protein (RhoGAP) that switches RhoA to its inactive form. We generated an Arhgap28-LacZ reporter mouse that revealed gene expression in soft tissues at E12.5, pre-bone structures of the limb at E15.5, and prominent expression restricted mostly to ribs and limb long bones at E18.5 days of development. Expression of recombinant Arhgap28-V5 in human osteosarcoma SaOS-2 cells caused a reduction in the basal level of RhoA activation and disruption of actin stress fibers. Extracellular matrix assembly studies using a 3-dimensional cell culture system showed that Arhgap28 was upregulated during Rho-dependent assembly of the ECM. Taken together, these observations led to the hypothesis that an Arhgap28 knockout mouse model would show a connective tissue phenotype, perhaps affecting bone. Arhgap28-null mice were viable and appeared normal, suggesting that there could be compensation from other RhoGAPs. Indeed, we showed that expression of Arhgap6 (a closely related RhoGAP) was upregulated in Arhgap28-null bone tissue. An upregulation in RhoA expression was also detected suggesting that Arhgap28 may be able to additionally regulate Rho signaling at a transcriptional level. Microarray analyses revealed that Col2a1, Col9a1, Matn3, and Comp that encode extracellular matrix proteins were downregulated in Arhgap28-null bone. Although mutations in these genes cause bone dysplasias no bone phenotype was detected in the Arhgap-28 null mice. Together, these data suggest that the regulation of Rho by RhoGAPs, including Arhgap28, during the assembly and development of mechanically strong tissues is complex and may involve multiple RhoGAPs.

Why are epididymal tumours so rare?

Epididymal tumour incidence is at most 0.03% of all male cancers. It is an enigma why the human epididymis does not often succumb to cancer, when it expresses markers of stem and cancer cells, and constitutively expresses oncogenes, pro-proliferative and pro-angiogenic factors that allow tumour cells to escape immunosurveillance in cancer-prone tissues. The privileged position of the human epididymis in evading tumourigenicity is reflected in transgenic mouse models in which induction of tumours in other organs is not accompanied by epididymal neoplasia. The epididymis appears to: (i) prevent tumour initiation (it probably lacks stem cells and has strong anti-oxidative mechanisms, active tumour suppressors and inactive oncogene products); (ii) foster tumour monitoring and destruction (by strong immuno-surveillance and -eradication, and cellular senescence); (iii) avert proliferation and angiogenesis (with persistent tight junctions, the presence of anti-angiogenic factors and misplaced pro-angiogenic factors), which together (iv) promote dormancy and restrict dividing cells to hyperplasia. Epididymal cells may be rendered non-responsive to oncogenic stimuli by the constitutive expression of factors generally inducible in tumours, and resistant to the normal epididymal environment, which mimics that of a tumour niche promoting tumour growth. The threshold for tumour initiation may thus be higher in the epididymis than in other organs. Several anti-tumour mechanisms are those that maintain spermatozoa quiescent and immunologically silent, so the low incidence of cancer in the epididymis may be a consequence of its role in sperm maturation and storage. Understanding these mechanisms may throw light on cancer prevention and therapy in general.

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor ß3.

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7days mediated by transforming growth factor (TGF) ß3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFß3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFß inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFß3 compared to MSCs. Therefore we added exogenous TGFß3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFß signaling or the addition of TGFß3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7days.

Norethisterone enanthate has neither a direct effect on the testis nor on the epididymis: a study in adult male cynomolgus monkeys (Macaca fascicularis).

OBJECTIVE: Norethisterone enanthate (NETE) is evaluated in trials of hormonal male contraception. It has been speculated that progestins may exert their contraceptive effects not only by suppressing gonadotropins but also by direct effects on male organs. NETE was given to monkeys in which endogenous gonadotropin secretion was suppressed by a gonadotropin releasing hormone (GnRH) antagonist, and replaced by human follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). If NETE has a direct effect on spermatogenesis and/or epididymal function, some changes in testicular histology, sperm motility and/or morphology should occur soon after exposure to NETE. METHODS: Fifteen adult intact male monkeys were grouped and treated for a 38-day period. Group I received GnRH antagonist, FSH, hCG and NETE while group II received a regime identical to group I without NETE and group III received only NETE and vehicle. Ejaculates, body weight, testicular biopsies and volume, and hormones were evaluated. RESULTS: There was a similar pattern of serum FSH and testosterone in groups I and II. Testicular volume and the proportion of tubuli exhibiting spermatids was significantly decreased in group III. There were no significant differences between group I and group II in any parameters measured. The forward progression of sperm was not affected by NETE treatment. The consistently low percentages of grade c sperm indicated no sign of hyperactivation. No changes in the gross morphology of the acrosome were detected. CONCLUSIONS: Short-term NETE treatment has neither a direct effect on the testis nor on the epididymis in this nonhuman primate model and its contraceptive effects appear to be exerted exclusively through gonadotropin suppression.

Mouse models of infertility due to swollen spermatozoa.

Transgenic mice with male infertility, the c-ros knockout (KO) and GPX5-Tag2 transgenic mouse models, are compared. Both exhibit severely angulated sperm flagella explaining the infertility. As angulated spermatozoa are swollen cells, a failure in volume regulation is indicated. Differences between genotypes were also found: caudal spermatozoa from c-ros KO, but not GPX5-Tag2, could fertilise eggs in vitro; flagellar angulation occurred more within the epididymis of GPX5-Tag2 than c-ros KO mice; the osmotic pressure of cauda epididymidal fluid was lower only in GPX5-Tag2 mice; angulation of caudal sperm from c-ros KO, but not GPX5-Tag2 mice, decreased upon demembranation. These observations indicate that GPX5-Tag2 mice express an earlier, more severe defect. Gene chip analyses of the epididymides revealed decreased expression of the CRES (cystatin-related epididymal-spermatogenic) and MEP17 (murine epididymal protein 17) genes in both genotypes. Further analysis could pinpoint genes essential for epididymal regulation of sperm volume, explain infertility and suggest modes of male contraception.

Increased luminal pH in the epididymis of infertile c-ros knockout mice and the expression of sodium-hydrogen exchangers and vacuolar proton pump H+-ATPase.

Transgenic mice targeted for the c-ros gene, which are fertile when heterozygous (HET), but infertile when homozygous (knockout, KO) and associated with failure in pubertal differentiation of the epididymal initial segment, provide a model for studying the role of the epididymal luminal environment in sperm development. Luminal fluid from the cauda epididymidis was measured by both ion-selective microelectrodes and pH strips to be 0.3 pH units higher in the KO than HET. Of the genes responsible for luminal acidification, expression of mRNA of vacuolar H(+)-ATPase was found in all epididymal regions, but with no difference between KO and HET. Immunohistochemistry showed its presence in epithelial apical cells and clear cells. The Na(+)-hydrogen exchanger NHE2 was expressed at mRNA and protein levels in the caput but only marginally detectable if at all in the distal epididymis. This was compensated for by NHE3 which was expressed strongest in the cauda region, in agreement with immunohistochemical staining. Quantification of Western blot data revealed slight, but significant, decreases of NHE2 in the caput and of NHE3 in the cauda in the KO mice. The increase in luminal fluid pH in the KO mice could also be contributed to by other epithelial regulating factors including the Na(+)-dependent glutamate transporter EAAC1 formerly reported to be down regulated in the KO.

Effects of putative epididymal osmolytes on sperm volume regulation of fertile and infertile c-ros transgenic Mice.

Volume regulation by spermatozoa has been demonstrated to be crucial in both mice and men for transport in the female tract. In order to determine the nature of osmolytes used by spermatozoa, they were released from the cauda epididymis of fertile c-ros heterozygous mice into incubation medium of uterine osmolality (representing an osmotic challenge), containing increasing concentrations of compounds that are major epididymal fluid components and known osmolytes in somatic cells. This should nullify the concentration gradients for osmolytes that mediate volume regulation, prevent osmolyte efflux, and lead to swelling. Of the osmolytes tested, K(+) caused the most rapid and extensive volume increases; glutamate, taurine, L-carnitine, and myo-inositol also were effective, but glycerophosphocholine was not. Such effects were not observed in cauda sperm from the infertile knockout mice, demonstrating a defect in normal volume regulation. K(+) concentrations in cauda epididymal fluid were 21 mM higher in the knockout than the heterozygous mice, but no differences were found in caudal fluid glutamate, carnitine, or myo-inositol. The carnitine content of cauda sperm from knockout males was not different from that of fertile males, but lower amounts of glutamate and inositol were found that could explain the poor volume regulation. In heterozygous mice, cauda but not caput sperm responded to the K(+) channel blocker quinine by swelling, demonstrating development of volume regulation during epididymal transit, whereas knockout cauda sperm showed no response, as with the osmolytes. Major epididymal secretions could serve as osmolytes in murine spermatozoa for volume regulation in response to physiological osmotic challenge in the normal fertile mice; the reduced sperm content of inositol and glutamate in the c-ros knockout mice might reflect maturational abnormalities in volume regulation.

Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice.

Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.

Sodium-inorganic phosphate cotransporter NaPi-IIb in the epididymis and its potential role in male fertility studied in a transgenic mouse model.

Analysis by cDNA microarrays showed that in the murine epididymis, NaPi-IIb was the predominantly expressed epithelial isoform of the sodium-inorganic phosphate cotransporter and was markedly overexpressed in the proximal region in the infertile knockout (KO) compared to the fertile heterozygous (HET) c-ros transgenic mouse. The apparent up-regulation in the KO mouse confirmed by Northern and Western blot analyses could be explained by the absence of NaPi-IIb from the initial segment of the HET epididymis, as revealed by immunohistochemistry, and its presence on the epithelial brush border throughout the proximal epididymis of KO mice, where differentiation of the initial segment fails to occur. Both NaPi-IIb mRNA and protein were scarce or absent from the cauda epididymidis of both genotypes. A high content of inorganic phosphate was measured enzymatically in the HET cauda luminal fluid, with a 27% decrease in the KO mice. This decrease, presumably from a greater reabsorption of inorganic phosphate, particularly in the initial part of the KO epididymis, may disturb the normal process of sperm maturation in these infertile males. By contrast, no apparent consequences were observed for the transport of Na+ and Ca2+, the concentrations of which (approximately 26 mM and approximately 30 microM, respectively) were measured by microelectrodes to be identical in the caudal fluid from both genotypes.

Epididymal dysfunction initiated by the expression of simian virus 40 T-antigen leads to angulated sperm flagella and infertility in transgenic mice.

We have generated two transgenic mouse lines (GPX5-Tag1 and GPX5-Tag2) by expressing the Simian virus 40 large and small T-antigens under a 5-kb promoter of the murine glutathione peroxidase 5 (GPX5) gene. In GPX5-Tag1 mice, with a high level of T-antigen expression, severe dysplasia was found in the epididymis and seminal vesicles. These mice also developed adrenal and prostate tumors, and spermatogenesis was disrupted. In GPX5-Tag2 mice, with a lower level of T-antigen expression, the only histological change was the slightly hyperplastic epithelium in the initial segment of the epididymidis and in the seminal vesicles. Despite normal mating behavior, these mice were infertile. The most conspicuous feature of the sperm was angulation of the flagellum, which appeared during epididymal transit, probably due to the observed reduction in the osmotic pressure of cauda epididymidal fluid. The angulation did not affect the motility or kinematic parameters of the sperm, but the sperm were also incapable of fertilization in vitro. The lack of expression of several genes specific for the initial segment suggests that in the GPX5-Tag2 mice the transgene expression brings about a differentiation arrest in this part of epididymis. This novel mouse line provides a model for epididymal dysfunction leading to defects in posttesticular sperm maturation and infertility.

Lack of glutamate transporter EAAC1 in the epididymis of infertile c-ros receptor tyrosine-kinase deficient mice.

Transgenic male mice carrying inactive mutations of the receptor tyrosine kinase c-ros lack the caput epididymidis initial segment and are infertile because sperm volume regulation is compromised. Complementary DNA arrays were used to detect differences in gene expression in the caput epididymidis of heterozygous fertile and homozygous infertile males. The glutamate transporter excitatory amino acid carrier 1 (EAAC1) was expressed in all epididymal regions with high expression in the initial segment and cauda epididymidis. Homozygous knockout mice did not express EAAC1 messenger RNA (mRNA) in the caput but they did express the gene in the corpus and cauda. Immunohistochemical staining for EAAC1 confirmed regional mRNA expression and demonstrated an adluminal location on stereocilia/microvilli of principal cells. The glutamate transporter-associated protein (GTRAP) 3-18 was detected in all epididymal regions independent of genotype, but a highly abundant novel transcript of 4.2 kilobases was found only in the initial segment of heterozygous c-ros mice. High-performance liquid chromatography measurement of glutamate revealed a significantly higher content in the proximal caput of infertile mice than fertile mice, and tissue glutamate content decreased distally in both genotypes. Because glutamate is used as an osmolyte in somatic cells, the lack of EAAC1 reported here may disturb normal osmolyte balance in the proximal epididymal lumen and compromise sperm maturation, in particular the development of sperm volume regulatory mechanisms.

Measurement of volume changes in mouse spermatozoa using an electronic sizing analyzer and a flow cytometer: validation and application to an infertile mouse model.

The importance of sperm volume has recently been highlighted in a knockout mouse model in which infertility was caused by defects in volume reguiation, which led to sperm transport failure in the female tract. Inhibition of volume regulation by human sperm, resulting in failure of penetration of cervical mucus in vitro, has also been reported. The present work aims to establish a sensitive and convenient method for monitoring changes in sperm volume for functional studies. Mature murine sperm obtained from the cauda epididymidis were analyzed by flow cytometry for their forward and side (90 degrees C) scatter of a 488-nm excitation wavelength laser, and the data were compared with volumes measured by electronic sizing using a Coulter counter. Changes in cell volume were induced by releasing or diluting sperm into culture media of various osmolalities (208-520 mmol/kg). Forward scatter signal (FSS) intensity correlated well with volume measurement obtained by a Coulter counter (R =.83; P <.001), confirming that FSS reflects Coulter counter findings as for somatic cells. Sperm swelling was also induced by the presence of quinine, a wide-spectrum channel blocker, in a medium of 330 mmol/kg, which is similar to the osmolality of uterine fluid. The effect of quinine on sperm volume was more obvious when analyzed by flow cytometry than by electronic sizing. This effect was even more marked after dead sperm identified by fluorescent dye were eliminated from analysis using flow cytometry. Swelling was characterized by an increase in forward scatter and side scatter, generating a subpopulation of sperm that correlated well (R =.79; P <.0001) with the population of sperm exhibiting an angulation of the tail, which is a morphological manifestation of swollen murine sperm. Flow cytometric analysis revealed that infertile sperm released from the cauda epididymidis of c-ros knockout mice were significantly larger than those of fertile sperm from heterozygous mice. This finding directly substantiates the suggestion that infertile sperm are defective in their volume regulation. Laser scatter analysis of viable murine sperm by flow cytometry offers a convenient and sensitive method for the study of sperm volume regulation.