Sperm Chromatin Status and DNA Fragmentation in Mouse Species with Divergent Mating Systems.

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus, and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

Sperm Capacitation and Kinematics in Phodopus Hamsters.

This study was designed to analyze changes in the spermatozoa of three species of Phodopus hamsters incubated under different conditions. Cauda epididymal sperm were incubated for 4 h in modified Tyrode's medium containing albumin, lactate, pyruvate, and Hepes (mTALP-H), in the same medium with the addition of bicarbonate (mTALP-BH), or with bicarbonate and 20 ng/mL of progesterone (mTALP-BH+P4). Media with bicarbonate are believed to promote capacitation in rodent species. Sperm motility, viability, capacitation patterns, and kinematics were assessed at different times. Capacitation in live cells was quantified after staining with Hoechst 33258 and chlortetracycline. Patterns believed to correspond to non-capacitated cells (F pattern), capacitated, acrosome-intact cells (B pattern), and acrosome-reacted cells (AR pattern) were recognized. Kinematics were examined via computer-assisted sperm analysis (CASA). The results showed a decrease in total motility in all three species in different media, with a sharp decrease in progressive motility in bicarbonate-containing media (without or with progesterone), suggesting hyperactivated motion. However, none of the other signs of hyperactivation described in rodents (i.e., decrease in STR or LIN, together with an increase in ALH) were observed. F pattern cells diminished with time in all media and were generally lower in P. roborovskii and higher in P. campbelli. B pattern cells increased in mTALP-BH media in all species. Progesterone did not enhance the percentage of B pattern cells. Finally, AR pattern cells increased in all species incubated in different media, showing the highest percentage in P. roborovskii and the lowest in P. campbelli. Comparisons between media revealed that there were higher percentages of F pattern cells and lower percentages of B pattern cells over time in medium without bicarbonate (mTALP-H) in comparison to media containing bicarbonate (mTALP-BH; mTALP-BH+P4). Overall, changes consistent with the acquisition of capacitation and development of hyperactivated motility were found; however, further studies are required to better characterize media necessary to support the pathways involved in these processes in Phodopus species.

Adenylate kinase 9 is essential for sperm function and male fertility in mammals.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

Effect of High Viscosity on Energy Metabolism and Kinematics of Spermatozoa from Three Mouse Species Incubated under Capacitating Conditions.

In order to sustain motility and prepare for fertilization, sperm require energy. The characterization of sperm ATP production and usage in mouse species revealed substantial differences in metabolic pathways that can be differentially affected by capacitation. Moreover, spermatozoa encounter different environments with varying viscoelastic properties in the female reproductive tract. Here, we examine whether viscosity affects sperm ATP levels and kinematics during capacitation in vitro. Sperm from three mouse species (Mus musculus, M. spretus, M. spicilegus) were incubated under capacitating conditions in a modified Tyrode's medium containing bicarbonate, glucose, pyruvate, lactate, and bovine serum albumin (mT-BH) or in a bicarbonate-free medium as a non-capacitating control. Viscosity was increased with the inclusion of polyvinylpyrrolidone. ATP was measured with a bioluminescence kit, and kinematics were examined with a computer-aided sperm analysis system. In M. musculus sperm, ATP declined during capacitation, but no differences were found between non-capacitating and capacitating sperm. In contrast, in M. spretus and M. spicilegus, ATP levels decreased in capacitating sperm. Increasing viscosity in the medium did not modify the timing or proportion of cells undergoing capacitation but did result in additional time- and concentration-dependent decreases in ATP in M. spretus and M. spicilegus under capacitating conditions. Additionally, increased viscosity altered both velocity and trajectory descriptors. The limited impact of capacitation and higher viscosity on M. musculus sperm ATP and kinematics could be related to the low intensity of postcopulatory sexual selection in this species. Responses seen in the other two species could be linked to the ability of their sperm to perform better under enhanced selective pressures.

Effect of Motility Factors D-Penicillamine, Hypotaurine and Epinephrine on the Performance of Spermatozoa from Five Hamster Species.

Assessments of sperm performance are valuable tools for the analysis of sperm fertilizing potential and to understand determinants of male fertility. Hamster species constitute important animal models because they produce sperm cells in high quantities and of high quality. Sexual selection over evolutionary time in these species seems to have resulted in the largest mammalian spermatozoa, and high swimming and bioenergetic performances. Earlier studies showed that golden hamster sperm requires motility factors such as D-penicillamine, hypotaurine and epinephrine (PHE) to sustain survival over time, but it is unknown how they affect swimming kinetics or ATP levels and if other hamster species also require them. The objective of the present study was to examine the effect of PHE on spermatozoa of five hamster species (Mesocricetus auratus, Cricetulus griseus, Phodopus campbelli, P. sungorus, P. roborovskii). In sperm incubated for up to 4 h without or with PHE, we assessed motility, viability, acrosome integrity, sperm velocity and trajectory, and ATP content. The results showed differences in the effect of PHE among species. They had a significant positive effect on the maintenance of sperm quality in M. auratus and C. griseus, whereas there was no consistent effect on spermatozoa of the Phodopus species. Differences between species may be the result of varying underlying regulatory mechanisms of sperm performance and may be important to understand how they relate to successful fertilization.

Influence of Different Regimes of Moderate Maternal Feed Restriction during Pregnancy of Primiparous Rabbit Does on Long-Term Metabolic Energy Homeostasis, Productive Performance and Welfare.

In this study, a maternal feed restriction (MFR; 105 g/d) in primiparous rabbit does was applied from day 0 to 7 post artificial insemination (AI) (R07, n = 96), from day 7 to 21 post AI (R721, n = 92), from day 0 to 21 post AI (R021, n = 94) or fed ad libitum during whole pregnancy (Control, n= 92). Feed intake (FI) was measured after MFR was over. On day 28 of gestation, fetoplacental development was evaluated (n = 11/group) and the productive parameters of the remaining dams were analyzed. Plasma free tri-iodothyronine (T3) and thyroxine, glucose, insulin, non-esterified fatty acids (NEFA), and corticosterone were analyzed during gestation and lactation (n = 5/group). After MFR, all groups significantly increased their voluntary FI. The longer MFR was, the lower the weight and length of the fetuses, but no long-term effects over litter performance were observed. R021 groups had the lowest T3 and the highest NEFA concentrations during pregnancy and showed insulin resistance at the end of gestation, but during lactation, energy homeostasis was balanced in all groups. MFR did not affect corticosterone concentrations. In conclusion, the ration setting applied slightly involved the energy homeostasis and metabolism of the animals, but their overall metabolic condition, productive performance and welfare were not compromised.

Gestation Food Restriction and Refeeding Compensate Maternal Energy Status and Alleviate Metabolic Consequences in Juvenile Offspring in a Rabbit Model.

Nutritional status during gestation can influence mother and offspring metabolism. Undernutrition in pregnancy affects women in both western and developing countries, and it is associated with a high prevalence of chronic diseases in later life. The present work was conducted in the rabbit model, as a longitudinal study, to examine the effect of food restriction during early and mid-gestation, and re-feeding ad libitum until the end of pregnancy on metabolic status and body reserves of mother and, its association with development and metabolism of fetuses and female offspring to the juvenile stage. Little changes in live body weight (LBW), compensatory feed intake, similar body reserves, and metabolism were observed in dams. Placenta biometry and efficiency were slightly affected, but fetal BW and phenotype were not modified. However, hyperinsulinemia, insulin resistance, and hypertriglyceridemia were demonstrated in pre-term fetuses. In the juvenile period, these changes were not evidenced, and a similar pattern of growth and serum metabolic parameters in offspring of food-restricted mothers were found, except in serum aminotransferases levels, which increased. These were associated with higher liver fibrosis. Maternal food restriction in the early and mid-pregnancy followed by re-feeding in our rabbit model established a compensatory energy status in dams and alleviated potential long-term consequences in growth and metabolism in the offspring, even if fetal metabolism was altered.

Physiological effects on rabbit sperm and reproductive response to recombinant rabbit beta nerve growth factor administered by intravaginal route in rabbit does.

Beta nerve growth factor (ß-NGF) is present in the seminal plasma of some species, including rabbits, acting as an ovulation-inducing factor in camelids. Traditionally, GnRH analogues are used to induce ovulation by intramuscular route when artificial insemination (AI) is performed in rabbit does. A specific rabbit recombinant ß-NGF (rrß-NGF) produced in our laboratory was tested as an alternative method to conventional treatment with GnRH analogues to induce ovulation. In the present work, different concentrations (0, 20, 100 ng/mL and 1, 20 and 100 µg/mL) of rrß-NGF were added to diluted semen to assess its effect on sperm traits (viability and motility parameters). rrß-NG was used also, incorporated to the AI dose, to evaluate ovulation response (LH and progesterone plasma concentrations, ovulation rate (OR) and embryo implantation at Day 7) after intravaginal administration. A negative control group stimulated with an empty catheter, and a positive control group inseminated and intramuscularly treated as usual with GnRH were also set up. Results showed that seminal quality was influenced by rrß-NGF depending on the concentration added, being the highest concentrations tested deleterious for semen. Whereas the highest OR was found in the positive control group (100%), concentrations of 20 ng/mL, 1 µg/mL and 20 µg/mL of rrß-NGF triggered intermediate OR (30, 60 and 42.9%, respectively), and 100 ng/mL and 100 µg/mL had the lowest OR (20 and 14.3%, respectively). Although LH peak was not observed in the first 2 h after AI in the ovulated females from rrß-NGF groups, plasma progesterone significantly increased at Day 7, except in those females treated with 20 and 100 µg/mL. Also, 98.4% of ovulated females were pregnant on Day 7. In conclusion, rrß-NGF added to diluted semen affects seminal quality and provokes ovulation, the development of functional CL and conception by intravaginal route in rabbit does, depending on the concentration added.

Intravenous immunoglobulin in macrophage activation syndrome associated with systemic lupus erythematosus. / Inmunoglobulina intravenosa en el síndrome de activación macrofágica asociado a lupus eritematoso sistémico.

BACKGROUND: The hemophagocytic lymphohistiocytosis is a disease with a potential fatal evolution, caused by the activation of macrophages and histiocytes with hemophagocytosis in bone marrow and other reticuloendothelial systems, triggered by a defect in the T lymphocyte when stimulating the production of interleukin 1-beta, interleukin 6, interferon-gamma and tumor necrosis factor-alpha that promote macrophage activation. This condition presents with fever, cytopenias, splenomegaly, hemophagocytosis in bone marrow, hypertriglyceridemia and hypofibrinogenemia, in the context of an infectious, neoplastic or autoimmune disease. The objective of this article is to describe the utility of intravenous immunoglobulin (IVIg) in patients unable to receive immunosuppressive treatment. CASE REPORTS: We present two case reports of systemic lupus erythematosus (SLE) with macrophage activation syndrome (MAS), one of them associated with central nervous system vasculitis and the other one with febrile neutropenia, both with bacterial infection added. CONCLUSIONS: The diagnosis of MAS should be suspected in all patients with lupus activity, fever, cytopenias, visceromegalies, hypertriglyceridemia and hypofibrinogenemia. Diagnosis and treatment are important to significantly reduce mortality. It is proposed that the first line treatment in patients that present SLE associated with MAS and sepsis should be IgIV and as a second line immunosuppressants with intravenous steroids.

INTRODUCCIÓN: La linfohistiocitosis hemofagocítica es una enfermedad de evolución potencialmente fatal, caracterizada por la activación de macrófagos y de histiocitos con hemofagocitosis en la médula ósea y en otros sistemas reticuloendoteliales, desencadenada por un defecto en los linfocitos T al estimular la producción de interleucina 1-beta, interleucina 6, interferón gamma y factor de necrosis tumoral alfa, que promueven la activación de los macrófagos. Esta afección cursa con fiebre, citopenias, esplenomegalia, hemofagocitosis en la médula ósea, hipertrigliceridemia e hipofibrinogenemia, en el contexto de una enfermedad infecciosa, neoplásica o autoinmunitaria. El objetivo de este artículo es describir la utilidad de la inmunoglobulina intravenosa (IgIV) en pacientes que no pueden recibir tratamiento inmunosupresor. CASOS CLÍNICOS: Se presentan dos casos de lupus eritematoso sistémico (LES) con síndrome de activación macrofágica (SAM), uno asociado a vasculitis del sistema nervioso central y otro a neutropenia febril, ambos con infección bacteriana agregada. CONCLUSIONES: El diagnóstico de SAM en pacientes con actividad lúpica se debe sospechar en caso de fiebre, citopenias, visceromegalias, hipertrigliceridemia e hipofibrinogenemia. Un diagnóstico y un tratamiento oportunos son importantes para disminuir de manera considerable la mortalidad. Se propone que el tratamiento de primera línea en los pacientes con LES asociado a SAM y sepsis sea la IgIV, y como segunda línea un inmunosupresor con un esteroide intravenoso.

Correction: Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits.

[This corrects the article DOI: 10.1371/journal.pone.0219780.].

Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits.

In some induced-ovulating species, beta nerve growth factor (ß-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant ß-NGF from rabbit tissue (rrß-NGF) to address the following objectives: 1) to compare rabbit ß-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrß-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrß-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rß-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to ß-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrß-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 µg/mL of the neuropeptide. Addition of this same concentration of rrß-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrß-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed.

Characterization of ß-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between ß-NGF and testosterone levels with seminal quality during sexual maturation.

ß-Nerve Growth Factor (ß-NGF) is a neurotrophin which acts through its receptors TrkA and p75, performing important actions in male reproductive physiology and its presence in seminal plasma (SP) has been related to male fertility. The aim of the present study was to evaluate the gene expression profile and the immunolocalization of ß-NGF and its high-affinity receptor TrkA in sex organs in rabbits during sexual maturation period. ß-NGF concentration for both SP and blood plasma (BP) and BP testosterone levels were determined as well as the seminal parameters during such period. Ten New Zealand White x California young rabbits were trained to semen collection since 20 weeks of age and routinely done once a week with two ejaculations per session. At 22 and 37 weeks of age, semen collection was carried out three times a week and seminal parameters were evaluated. Four males were randomly assigned and slaughtered in each age (n = 8); sex organs (prostate, bulbourethral glands and epididymis) were dissected and collected to determine ß-NGF and TrkA gene expression and immunolocalization. SP and BP were also taken at each semen collection session to evaluate ß-NGF concentration, and testosterone levels were also assessed in BP. The highest ß-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual ß-NGF mRNA expression and limited localization of the neurotrophin. The prostate epithelial cells and lumen were strongly stained with regard to the other sex organs indicating that immunolocalization of ß-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, ß-NGF concentration in SP and BP remained unchanged in accordance to age, while some seminal characteristics such as sperm concentration, percentage of live sperm and mass and progressive motility were enhanced as endowed by BP testosterone variation. ß-NGF and its cognate TrkA receptor are expressed and immunolocalized in the male reproductive tract in the two ages studied, independently of the circulating levels of testosterone and ß-NGF.

Gene expression and immunolocalization of low-affinity neurotrophin receptor (p75) in rabbit male reproductive tract during sexual maturation.

This study reports the gene expression and immunolocalization of the low-affinity neurotrophin receptor, p75, in accessory glands (prostate and bulbourethral glands) and epididymis (caput and cauda) of male rabbits during sexual maturation. We showed that p75 was expressed in all tissues studied with similar mRNA levels during this period. However, it was differentially immunolocalized in bulbourethral glands and stereocilia of epididymis. These findings may be related with some remodeling processes in the accessory glands during sexual maturation, which could be associated with sperm maturation. The interaction of p75 with neurotrophins in rabbit male reproductive tract suggests the possible implication of this system in sexual maturation in rabbits.

Safety assessment on pedestrian crossing environments using MLS data.

In the framework of infrastructure analysis and maintenance in an urban environment, it is important to address the safety of every road user. This paper presents a methodology for the evaluation of several safety indicators on pedestrian crossing environments using geometric and radiometric information extracted from 3D point clouds collected by a Mobile Mapping System (MMS). The methodology is divided in four main modules which analyze the accessibility of the crossing area, the presence of traffic lights and traffic signs, and the visibility between a driver and a pedestrian on the proximities of a pedestrian crossing. The outputs of the analysis are exported to a Geographic Information System (GIS) where they are visualized and can be further processed in the context of city management. The methodology has been tested on approximately 30 pedestrian crossings in cluttered urban environments of two different cities. Results show that MMS are a valid mean to assess the safety of a specific urban environment, regarding its geometric conditions. Remarkable results are presented on traffic light classification, with a global F-score close to 95%.

Equine viral arteritis in breeding and sport horses in central Spain.

Equine viral arteritis (EVA) may have a high economic impact on breeding stud farms due to the occurrence of EVA-associated abortion outbreaks and the ability of the virus to persist in carrier stallions. While the consequences of EVA in premises with sport horses are usually less severe, the first confirmed outbreak of EVA in Spain occurred in a riding club in Barcelona, but no data on the seroprevalence of EVA in sport horses have been reported in Spain. Given the importance of both Spanish Purebred (SP) breeding horses and sport horses for Spain's equine industry, the aim of this study was to determine and compare the seroprevalence of EVA in these two horse populations in central Spain. Serum samples from 155 SP breeding horses residing in 16 stud farms and 105 sport horses of different breeds housed in 12 riding clubs, collected between September 2011 and November 2013, were tested using a commercial EVA antibody ELISA test with a 100% sensitivity, and confirmed by seroneutralisation (SN) test. EVA seroprevalence in SP breeding horses was higher 21.1% (95% CI 15.3-26.8%) than that in sport horses (6.7%, 95% CI 1.89-11.45%). However, the primary use (breeding vs. sport) was not significantly associated with seropositivity to Equine Arteritis Virus (EAV), suggesting that different management factors do not affect EVA circulation in these two horse populations.

Sex and cohort as sources of differentiation of values in postmodern society

The aim of this study was to analyze sex and cohort differences in ranking-emphasis on values measured using the Rokeach Value Survey in two groups of 122 adult and 150 young participants. Results indicate differences between cohorts in two-thirds of the final values and half of the instrumental values, and sex differences in seven final values and four instrumental values, showing a change in relevant values mostly in females. The results are discussed from the perspective of socio-cultural change that has taken place in Spain in the last decades and from the perspective of change in values in postmodern western society (AU)

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