A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture.

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.

The reproductive success of bovine sperm after sex-sorting: a meta-analysis.

In the three decades since its inception, the sex-sorting technology has progressed significantly. However, field studies report conflicting findings regarding reproductive outcomes. Therefore, we conducted this meta-analysis of all trials published between 1999 and 2021. Non-return rates after 24 or 60 d (NRR 24/60), pregnancy, calving, abortion, and stillbirth rates were compared after AI with sex-sorted vs non-sorted sperm. Additionally, the impact of recent developments in the sex-sorting technology was assessed. Of 860 studies found, 45 studies (72 trials) provided extractable data and were included. Overall, the results of this meta-analysis provided evidence that the NRR 24/60 was diminished by 13%, pregnancy rates were reduced by 23% (25% cows, 21% heifers) and calving rates were reduced by 24% when using sex-sorted sperm. Enhancing the dosage to 4 million sex-sorted sperm/straw (including recent improvements, high vs low dose) as well as using fresh sex-sorted sperm (sorted vs non-sorted) increased pregnancy rate ratios by 7 percentage points. The refinement of the sex-sorting technology after 2015 resulted in a lowered reduction of pregnancy and calving rate of 19% and 23%, respectively. Whereas abortion rates were similar, the stillbirth of male calves was increased by 6.3%.

Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting.

To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.

Early embryo-maternal communication in the oviduct: A review.

An intact embryo-maternal communication is critical for the establishment of a successful pregnancy. To date, a huge number of studies have been performed describing the complex process of embryo-maternal signaling within the uterus. However, recent studies indicate that the early embryo communicates with the oviductal cells shortly after fertilizationand that this is important for the successful establishment of pregnancy. Only if the early embryo is capable to signal the mother within a precise timeframe and to garner a response, will the embryo be able to survive and reach the uterus. This review will give an overview of all the experimental designs which have investigated embryo-maternal interaction in the oviduct. In addition to that, it will provide a comprehensive analysis of the findings to date elucidating the morphological and molecular changes in the oviduct which are induced by the presence of the early embryo highlighting how the tubal responses affect embryo development and survival.

Exploring patient preference regarding interpreter use in primary care.

BACKGROUND: Effective communication is considered an essential component of delivering health care. Trained, professional interpreters are the gold standard for overcoming language barriers with those with limited English proficiency (LEP). However, LEP patients often use unqualified interpreters such as family members and friends. Existing literature explores the rationale behind choosing different interpreters, but rarely from the patient perspective. AIM: To explore the patient perspective on the type of interpreter best suited for primary care consultations. METHOD: Participants self-identified as having LEP were recruited from four GP practices in areas of Sheffield with high proportions of black and minority ethnic (BME) residents. The participants were from Urdu-, Arabic-, or Romani-speaking ethnic groups. Semi-structured interpreted interviews were recorded, transcribed, and analysed thematically with independent verification of emergent themes. Interviews continued to data saturation. RESULTS: All participants expressed a preference for face-to-face interpreters. Urdu and Arabic participants highlighted the importance of using an interpreter with the same dialect; Roma participants were passionate about the need for qualified Roma interpreters. Most participants also identified trust and sex as important factors. However, interpreter preference varied between participants: some valued the continuity of family members, whereas others favoured the professionalism and linguistic accuracy associated with qualified interpreters. CONCLUSION: This study identified conflicts between patient preferences and guidance for healthcare professionals; all of the participants disliked telephone interpreting, and many recognised the benefits of untrained interpreters. The study highlights the complexities of interpreter preference in primary care and suggests that the decision should be flexible, and patient centred.

Key attributes of global partnerships in food and nutrition to align research agendas and improve public health.

Partnerships among academia, government, and industry have emerged in response to global challenges in food and nutrition. At a workshop reviewing international partnerships, we concluded that to build a partnership, partners must establish a common goal, identify barriers, and engage all stakeholders to ensure project sustainability. To be effective, partnerships must synchronize methodologies and adopt evidence-based processes, and be led by governmental or nonprofit organizations to ensure trust among partners and with the public.

Mucocutaneous paraneoplastic syndromes associated with hematologic malignancies.

Cutaneous paraneoplastic syndromes are a group of dermatoses that demonstrate a range of morphological and pathological findings. These syndromes may precede, be concurrent with, or follow the diagnosis of an underlying malignancy. Treatment of the malignancy is often associated with improvement in or resolution of the mucosal and cutaneous disease; however, this is not the case with paraneoplastic pemphigus (PNP). PNP is a rare syndrome that was first described in 1990, and it occurs almost exclusively in patients with lymphocytic neoplasms. Pulmonary manifestations occur in 30% to 40% of cases, and it is the only form of pemphigus that attacks epithelium other than squamous epithelium in an antibody-mediated fashion. The mortality rate for PNP associated with malignancy is greater than 90%. Treatment guidelines are not available, but case series point to the use of rituximab (Rituxan) as well as corticosteroids and various other immunomodulating agents. Here we present a diagnostic and treatment dilemma in a 39-year-old active-duty male who developed PNP in the setting of treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for grade 3 follicular lymphoma. This case report is followed by a review of the diagnosis and treatment of other cutaneous paraneoplastic syndromes that are associated with hematologic malignancies.

Solventless sampling of phthalate esters.

An environmentally friendly method to passively sample six phthalate esters in water has been devised. The sampling device consists of a semi-permeable membrane, which the analytes diffuse through and are collected onto Tenax TA. The Tenax TA was then thermally desorbed with a helium stream into a gas chromatograph-mass spectrometer (GC-MS) after the sampling period. Samplers were exposed to water samples for known periods of time at known concentrations to create time-weighted average (TWA) plots. For all six phthalates the resulting TWA plots had linear correlations, allowing for the determination of permeation constants for the phthalates. The lag time for each phthalate was also determined using this method. An advantage of this method is that no solvents are used. The analytes are removed from the Tenax TA using thermal desorption rather than liquid extraction.

Permeation sampling of phthalate esters in water.

A method for sampling the six phthalate esters considered priority pollutants by the United States EPA has been developed. The sampling device utilizes a silicone polycarbonate membrane (SSP-M213) through which the analytes permeate and are collected onto an adsorbent, Amberlite XAD-16. After a known period, the samplers were removed from the water and the analytes were extracted from the adsorbent using dichloromethane/hexane (50:50, v/v), with the resulting solution analyzed using gas chromatography with flame ionization detection. Time-weighted average (TWA) concentrations of the phthalates in the water were determined by creating plots of amount of analyte collected versus the product of concentration and time (ppm. hr) for each phthalate. A linear correlation between the amount of analyte collected and the product of concentration and time was seen for each phthalate. Effects of temperature, stirring rate and the presence of potential interferents on the rate of permeation were also studied. The obtained and minimal amounts of solvent are used for sample preparation and injection into the gas chromatograph.

Effects of MHC and gender on lupus-like autoimmunity in Nba2 congenic mice.

The lupus-like disease that develops in hybrids of NZB and NZW mice is genetically complex, involving both MHC- and non-MHC-encoded genes. Studies in this model have indicated that the H2d/z MHC type, compared with H2d/d or H2z/z, is critical for disease development. C57BL/6 (B6) mice (H2b/b) congenic for NZB autoimmunity 2 (Nba2), a NZB-derived susceptibility locus on distal chromosome 1, produce autoantibodies to nuclear Ags, but do not develop kidney disease. Crossing B6.Nba2 to NZW results in H2b/z F1 offspring that develop severe lupus nephritis. Despite the importance of H2z in past studies, we found no enhancement of autoantibody production or nephritis in H2b/z vs H2b/b B6.Nba2 mice, and inheritance of H2z/z markedly suppressed autoantibody production. (B6.Nba2 x NZW)F1 mice, compared with MHC-matched B6.Nba2 mice, produced higher levels of IgG autoantibodies to chromatin, but not to dsDNA. Although progressive renal damage with proteinuria only occurred in F1 mice, kidneys of some B6.Nba2 mice showed similar extensive IgG and C3 deposition. We also studied male and female B6.Nba2 and F1 mice with different MHC combinations to determine whether increased susceptibility to lupus among females was also expressed within the context of the Nba2 locus. Regardless of MHC or the presence of NZW genes, females produced higher levels of antinuclear autoantibodies, and female F1 mice developed severe proteinuria with higher frequencies. Together, these studies help to clarify particular genetic and sex-specific influences on the pathogenesis of lupus nephritis.

Activation of carbonic anhydrase II by active-site incorporation of histidine analogs.

The hydration of CO2 catalyzed by human carbonic anhydrase II (HCA II) is accompanied by proton transfer from the zinc-bound water of the enzyme to solution. We have replaced the proton shuttling residue His 64 with Ala and placed cysteine residues within the active-site cavity by mutating sites Trp 5, Asn 62, Ile 91, and Phe 131. These mutants were modified at the single inserted cysteine with imidazole analogs to introduce new potential shuttle groups. Catalysis by these modified mutants was determined by stopped-flow and 18O-exchange methods. Specificity in proton transfer was demonstrated; only modifications of the Cys 131-containing mutant showed enhancement in the proton transfer step of catalysis compared with unmodified Cys 131-containing mutant. Modifications at other sites resulted in up to 3-fold enhancement in rates of CO2 hydration, with apparent second-order rate constants near 350 microM(-1) s(-1). These are among the largest values of kcat/Km observed for a carbonic anhydrase.

Direct atmospheric pressure coupling of polyacrylamide gel electrophoresis to mass spectrometry for rapid protein sequence analysis.

Using laser desorption-atmospheric pressure chemical ionization we describe a novel approach for coupling mass spectrometry to polyacrylamide gel electrophoresis. In contrast to other approaches, the method allows for the direct sampling of a polyacrylamide gel-embedded protein without the addition of any exogenous matrixes and is performed at atmospheric pressure. After electrophoresis and enzymatic digestion, the gel is analyzed at AP by photons that desorb neutral peptide molecules, followed by corona discharge ionization in the gas-phase, and subsequent mass analysis. Our experimental results demonstrate the method to (1) rapidly identify electrophoresed proteins via "peptide fingerprinting" using protein databases, (2) detect single-amino acid polymorphisms, and (3) has potential for sub-picomole sensitivity while still maintaining in situ gel desorption-ionization at ambient conditions.

Laser desorption-atmospheric pressure chemical ionization: a novel ion source for the direct coupling of polyacrylamide gel electrophoresis to mass spectrometry.

Laser desorption-atmospheric pressure chemical ionization-mass spectrometry (LD-APCI-MS) is presented for the atmospheric pressure (AP) sampling of tryptic peptides directly from a polyacrylamide gel. In contrast to other gel sampling mass spectrometric approaches, this technique does not require the addition of any exogenous matrices to the gel to assist with ionization. In this arrangement, a CO(2) laser at 10.6 micro m is used to desorb intact neutral peptide molecules from the gel, followed by ionization in the gas-phase with APCI. The ions are then sampled via a heated capillary inlet and transferred to a quadrupole ion trap mass spectrometer for mass analysis. Preliminary results suggest the polyacrylamide gel electrophoresis-LD-APCI-MS technique provides several advantages that could translate into a more convenient, robust methodology for the rapid identification and characterization of proteins. Finally, strategies regarding the further development of the method are presented.