Sperm proteins ODF2 and PAWP as markers of fertility in breeding bulls.

Low fertility is the single most important factor limiting livestock reproductive performance, adversely affecting the cattle industry and causing millions of dollars of economic loss. In the livestock industry, male fertility is of crucial importance for the reproductive performance of livestock. However, there is a lack of reliable biomarkers to predict bull fertility in artificial insemination service. The objective of this study was to identify sperm proteins as biomarkers for bull fertility. To discover candidate sperm quality biomarkers, sperm proteome profiling was conducted in extreme high- and extreme low-fertile bulls selected from a pool of 1000 AI sires with varied fertility. Thirty-two differentially expressed proteins were identified. Among them, high levels of sperm outer dense fiber of sperm tails 2 (ODF2) and post-acrosomal assembly of sperm head protein (PAWP/WBP2NL) represented the most extreme differences in quantity between high- and low-fertility bulls. Protein immunodetection and flow cytometry used to validate these putative fertility markers in a combined cohort of 154 AI sires. Both ODF2 and PAWP correlated significantly with fertility. In conclusion, ODF2 and PAWP can be used to assess semen quality and predict sire fertility.

Comparison of two culture media on morphokinetics and ploidy status of sibling embryos.

To investigate the effects of culture media with different lactate concentrations on early embryonic development, data collected from our patients undergoing preimplantation genetic testing (PGT) were assessed using the EmbryoScope™ time-lapse culturing system. After intracytoplasmic sperm injection (ICSI), sibling oocytes were cultured in the same EmbryoScope (Vitrolife) slides including two different commercially available media. The patients with fewer than five mature oocytes were not included in the analyses. All embryos were hatched on day 3, and trophectoderm biopsies (n = 212) were performed accordingly. PGT for aneuploidy (PGT-A) on biopsied materials was carried out using next generation sequencing. Morphokinetic parameters, fertilization, irregular division, degeneration, blastulation, euploidy, and pregnancy rates of embryos cultured in LifeGlobal Global Total medium (LGGT) and Continuous Single Culture-NX Complete medium (CSCM-NXC) were compared. There were no differences observed in time to pronuclear fade, or in time spent as 2-cell (cc2) and 3-cell (s2), to 4-cell, 5-cell, morula and blastocyst stages (P > 0.05). Embryos reached the 2-cell (t2) and 3-cell (t3) stages significantly faster in LGGT (P < 0.05), whereas embryos grown in CSCM-NXC with lower lactate reached starting blastulation significantly sooner (P = 0.026). However, there were no statistical differences observed in fertilization, blastulation, degeneration, irregular division euploidy, and pregnancy rates between the two groups (P > 0.05). Even though pregnancy and fertilization rates did not indicate statistical differences, results are significant to provide better insight on potential roles of lactate in embryo development. These finding will advance the fundamental knowledge of human embryo development and assisted reproductive technologies.

Coculture model of blood-brain barrier on electrospun nanofibers.

The blood-brain barrier (BBB) is a control mechanism that limits the diffusion of many substances to the central nervous system (CNS). In this study, we designed an in-vitro 3-dimensional BBB system to obtain a fast and reliable model to mimic drug delivery characteristics of the CNS. A support membrane of polycaprolactone nanofiber surfaces was prepared using electrospinning. After confirming the fiber morphology and size, endothelial cells (HUVEC) and glial cells were cultured on either side of this membrane. The model's similarity to in vivo physiology was tested with a home-designed transmembrane resistance (TR) device, with positive and negative control molecules. Finally, 2 doses of methotrexate (MTX), a chemotherapy agent, were applied to the model, and its permeability through the model was determined indirectly by a vitality test on the MCF-7 cell line. Nicotine, the positive control, completed its penetration through the model almost instantly, while albumin, the negative control, was blocked significantly even after 2 days. MTX reached a deadly threshold 24 h after application. The TR value of the model was promising, being around 260 ohm.cm2. The provided model proposes a disposable and reliable tool for investigating drug permeability through the BBB and has the potential to reduce the number of animal experiments.

The long-term protective effect of hypothermia and gradual detorsion on ovarian tissue in adnexal torsion/detorsion model in rats.

INTRODUCTION: Ischemia in ovarian torsion and subsequent reperfusion has significant effects on fertility in the long term. The most important reason for these changes is thought to be a reperfusion injury rather than ischemia. We aimed to evaluate whether the reperfusion injury following ovarian detorsion could be reduced by hypothermia and intermittent reperfusion. MATERIALS AND METHODS: Forty adult female rats were divided into five groups as follows: Sham (Sh) (n = 8), torsion detorsion (control TD) (n = 8), progressive reperfusion "gradual detorsion" (GD) (n = 8), hypothermia (H) (n = 8) and the progressive reperfusion + hypothermia (GD + H) (n = 8). In all rats, except for the Sh group, the left ovary was rotated counter clockwise 1080° and fixed to the abdominal wall by three 5-0 non-absorbable sutures followed by the closure of the laparotomy. After 30 h, reperfusion was achieved following the detorsion of the ovaries. In both the control TD and H groups, the torsed ovaries were detorsed. H group, however, was subjected to hypothermia with ice packs 30 min before and during the detorsion. Tissue temperature was kept constant at 4 °C, controlled by a digital thermometer. In the GD group, the torsed ovary and pedicle were detorsed by 360°, followed by a 5 min pause. This procedure was repeated twice until a complete detorsion was achieved. GD + H group underwent hypothermia with ice packs 30 min before the procedure and the torsed ovary and pedicle were detorsed by 360°. After a 5 min pause, we repeated this process twice to provide full detorsion. The tissue temperature was constantly held at 4 °C. In the hypothermia groups, we applied hypothermia for an additional 30 min after detorsion and then left the rats at normal body temperature during reperfusion. We followed the rats in all groups for 60 days. Then we excised the left ovaries of all rats through laparotomy and spared some of the ovaries for biochemical and pathological examination. Intracardiac blood was taken at the end of the procedure and it was sent to the biochemical laboratory to assess oxidative stress markers. Finally, all the animals were sacrificed with high-dose of anesthesia. RESULTS: Evaluation of the results revealed that oxidative stress markers were significantly lower, and antioxidant parameters were higher in the experimental groups compared with the control TD group (p < 0.05). Histopathologically, we found that tissues were preserved in GD, H, GD + H groups (p < 0.05). When we compared the groups among each other, both biochemical and histopathological values in GD + H group showed that the tissue was preserved from oxidative damage, albeit the difference did not reach a level of significance. DISCUSSION: Several studies have shown that both hypothermia and intermittent reperfusion protect tissue from IR damage in the early period. However, as far as we know there is no study on long-term outcomes of both practices. Our study showed that both hypothermia and intermittent reperfusion alone protect tissue from IR damage in the long term. However, it did not show the superiority of the combination of both methods compared to that of individual application. The advantages of these methods lie in their easy application and cost-effectiveness. We believe that our study will serve as a base for future studies on the subject.

Anti-quorum sensing and anti-biofilm activities of Hypericum perforatum extracts against Pseudomonas aeruginosa.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. (Hypericaceae) has been used as a traditional therapeutic for skin wounds, burns, cuts and stomach ailments including stomach ache, ulcers for a long time in many societies. Although many studies about its antibacterial properties can be found, there is a lack of studies about its quorum sensing inhibition properties, which effects bacterial vulnerability directly, on Pseudomonas aeruginosa. AIM OF THE STUDY: Evaluation of anti-quorum sensing (anti-QS) and anti-biofilm activity of ethanol, methanol, acetone and ultra-sonicated extracts of Hypericum perforatum L. (HP) which is a well-known wound healer, against P. aeruginosa. MATERIALS AND METHODS: Aerial parts of HP were extracted with ethanol, methanol and acetone. In addition, separate extractions with ultrasonication were carried out with same solvents. Anti-QS activity tests with different doses of HP extracts were performed by employing biomonitor strains, of which the promoter of QS regulating and green fluorescent protein (GFP) genes were fusioned. For anti-biofilm activity, HP extracts were applied to wild type PAO1 strains and biofilm inhibition was quantified via crystal violet staining method. RESULTS: HP's ethanol, methanol and acetone extracts (250 µg/ml doses) inhibited LasIR signalling pathway up to 65.43%, 59.60%, 55.95% and same solvent extracts obtained with ultrasonication inhibited 71.33%, 64.47%, 57.35% respectively. Moreover, inhibition rates of RhlIR pathway were 28.80%, 50.83%, 45.84% for ethanol, methanol, acetone extracts (250 µg/ml doses) and 51.43%, 57.41%, 50.02% for ultrasonication extracts (250 µg/ml doses), compared to untreated controls. In the experiments, ethanol, methanol, acetone and ultra-sonicated extracts of HP did not inhibit biofilm formation. CONCLUSIONS: This study shows that HP plant is capable for blocking of las and rhl QS systems of P. aeruginosa. However, it was observed that ethanol, methanol and acetone extract of the plant samples did not show anti-biofilm activity against P. aeruginosa. This led us to thinking that biofilm formation was caused via another pathway such as IQS or PQS. Further studies with isolated active compounds of HP might give a better understanding of the effects on biofilm formation of P. aeruginosa.

Sperm protamine-status correlates to the fertility of breeding bulls.

During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.

Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility.

Sub-par fertility in bulls is influenced by alterations in sperm chromatin, and it might not be solved with increased sperm concentration in artificial insemination. Appropriate histone retention during sperm chromatin condensation plays critical roles in male fertility. The objective of this study was to determine failures of sperm chromatin condensation associated with abnormal persistence or accessibility of histones by aniline blue (ANBL) test, expression levels, and cellular localizations of one variant and two core histones (H3.3, H2B, and H4 respectively) in the spermatozoa of low-fertility (LF) vs high-fertility (HF) bulls. The expression levels and cellular localizations of histones in spermatozoa were studied using immunoblotting, immunocytochemistry, and staining methods. The bioinformatics focused on the sequence identity and evolutionary distance of these proteins among three mammalian species: bovine, mouse, and human. We demonstrated that ANBL staining was different within the LF (1.73 (0.55, 0.19)) and HF (0.67 (0.17, 0.06)) groups (P<0.0001), which was also negatively correlated with in vivo bull fertility (r=-0.90, P<0.0001). Although these histones were consistently detectable and specifically localized in bull sperm cells, they were not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and were evolutionarily conserved in bulls, mice and humans. The H2B variants were more conserved between bulls and humans, than in mice. In conclusion, we showed that H2B, H3.3, and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related to bull fertility.

Interrelationships between apoptosis and fertility in bull sperm.

Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = -0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = -0.49, P<0.006 and r = -0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1-3 vs. Bull 4-5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.

Delivering value from sperm proteomics for fertility.

Fertilization of an egg by a spermatozoon sets the stage for mammalian development. Viable sperm are a prerequisite for successful fertilization and beyond. Spermatozoa have a unique cell structure where haploid genomic DNA is located in a tiny cytoplasmic space in the head, mitochondria in the midpiece and then the tail, all enclosed by several layers of membrane. Proteins in sperm play vital roles in motility, capacitation, fertilization, egg activation and embryo development. Molecular defects in these proteins are associated with low fertility or in some cases, infertility. This review will first summarize genesis, molecular anatomy and physiology of spermatozoa, fertilization, embryogenesis and then those proteins playing important roles in various aspects of sperm physiology.

IVM is an alternative for patients with PCO after failed conventional IVF attempt.

PURPOSE: To determine if IVM of oocytes from unstimulated cycle is a treatment option for patients who did not deliver after standard IVF-ET. METHOD: Twenty three women with PCO, thirteen of them with normal cycles and all <35 years old, who failed IVF served as their own control. During the control IVF cycle patients were stimulated with 1730.7 ± 639.5 IU recombinant FSH, a long Buserelin acetate protocol was used and embryo transfer was performed on day 2 or 3 after ICSI. After failed IVF immature oocytes were aspirated transvaginally from antral follicles during spontaneous menstrual cycle. Embryo transfer was performed 2 or 3 days later. RESULT: 11.4 ± 4.8 mature oocytes and 6.7 ± 3.2 embryos were produced with IVF, which served as the control, compared to 9.7 ± 4.5 mature oocytes and 6.2 ± 3.2 embryos with IVM. There was one clinical pregnancy in the IVF group which did not result in a live birth where as five singleton and one pair of twins with healthy live births and one miscarriage in the IVM group. CONCLUSION: IVM does not involve ovarian stimulation with possible financial and health consequences. It may be an useful treatment after unsuccessful IVF.