Epididymal sperm quality of Kacang goat preserved in low temperature for genetic material utilization in assisted reproductive technologies

Postmortem epidydimal preservation at low temperature (3-4°C), is a way to preserve and recover male genetic material. This effort aims for prolonging male function as sperm source, followed with its utilization in assisted reproductive technologies. This study aimed to observe the quality of sperm form cauda epididymis which preserved at low temperature for consecutive days. Sperm were retrieved from twelve cauda epididymis of Kacang Goat and its qualities namely motility, intact membrane, life/dead, and abnormality (all in %) were evaluated in every 2 days until 0% motility. Data were compared using analysis of variance at a = 0.05. Result shows significant (P<0.05) decrease in motility, intact membrane, and life/dead, but increase in abnormality during observation at day 0, day 2, day 4 and day 6, respectively. At the respective days, motility was 91.33±1.25%; 74.67±3.88%; 28.17±2.25% and 0.33±0.57%, intact membrane was 54.83±1.04%; 39±3.77%; 25.1±3.32% and 14.83±2.75%, life/dead was 55.17±4.01%; 36±3.5%; 24.3±3.25% and 12±2.78%, abnormality was 3.16±0.76%; 4.16±0.76%; 6.16±2.25% and 11±2.17%. According to the study, it is concluded that preserved sperm from cauda epididymis at low temperature shows decrease in quality and its utilization should rely on the quality status to select the most appropriate assisted reproductive technology.

Mechanosensitive channel YnaI has lipid-bound extended sensor paddles

The general mechanism of bacterial mechanosensitive channels (MS) has been characterized by extensive studies on a small conductance channel MscS from Escherichia coli (E. coli). However, recent structural studies on the same channel have revealed controversial roles of various channel-bound lipids in channel gating. To better understand bacterial MscS-like channels, it is necessary to characterize homologs other than MscS. Here, we describe the structure of YnaI, one of the closest MscS homologs in E. coli, in its non-conducting state at 3.3 Å resolution determined by cryo electron microscopy. Our structure revealed the intact membrane sensor paddle domain in YnaI, which was stabilized by functionally important residues H43, Q46, Y50 and K93. In the pockets between sensor paddles, there were clear lipid densities that interact strongly with residues Q100 and R120. These lipids were a mixture of natural lipids but may be enriched in cardiolipin and phosphatidylserine. In addition, residues along the ion-conducting pathway and responsible for the heptameric assembly were discussed. Together with biochemical experiments and mutagenesis studies, our results provide strong support for the idea that the pocket lipids are functionally important for mechanosensitive channels.

Cervical ureaplasma colonization affects intraamniotic inflammation in preterm labor with intact membrane: a cohort study

Background: A causative role between cervical ureaplasma colonization and adverse outcomes during pregnancy has remained controversial. We investigated whether cervical ureaplasma colonization affects the biochemically or histologically intraamniotic inflammation in preterm birth.Methods: Amniotic fluid was retrieved during delivery. Various chorioamnionitis-related cytokines (interleukin (IL)-1β, -6, -8, -10, and tumor necrosis factor-α) and regulators (matrix metalloproteins (MMP)-8 and MMP-9) were measured with Human Magnetic Luminex screening assay. We tested cervical swab specimens using real-time polymerase chain reaction assays for the detection of ureaplasma spp. colonization. Considering the clinical situation that causes intraamniotic infection, we arbitrarily divided into three categories of preterm labor with intact membrane, preterm premature rupture of membrane (PPROM), and control group with no exposure to preterm labor or preterm premature rupture of membrane.Results: The incidence of cervical ureaplasma colonization was 49.3% (136/276). The incidence of histologic chorioamnionitis was 27.5% (76/200). All differences in cytokines and regulators according to histologic chorioamnionitis were significant. Of the 153 cases that experienced preterm labor with intact membrane, IL-10, MMP-8, and MMP-9 levels in the ureaplasma positive group were significantly higher than those of the ureaplasma negative group. According to logistic regression analysis adjusted to preterm labor with intact membrane, PPROM, and gestational age at delivery, cervical ureaplasma colonization was an independent risk factor of histologic chorioamnionitis (odd ratio: 2.622, 95% confidence interval: 1.443-4.766).Conclusions: Cervical ureaplasma colonization augments biochemically intraamniotic inflammation in preterm labor with intact membrane, and was an independent risk factor of histologic chorioamnionitis.

Influence of Non-conventional Sperm Quality Parameters on Field Fertility in Ovine

The prediction of the fertilizing ability of a seminal dose continues to be a primary aim in the field of artificial insemination (AI). To achieve this goal, in this study we have included the evaluation of some non-conventional sperm quality markers. A total of 3,906 ewes from 52 different farms were inseminated with 357 refrigerated seminal doses obtained from 45 mature Rasa Aragonesa rams. The same samples were used for sperm quality analysis including membrane integrity, capacitation status, oxygen consumption and apoptotic-like markers such as phosphatidylserine translocation (PS), plasmalemma disorganization/mitochondrial membrane potential, caspase activation and DNA damage. Seminal doses from the breeding (B) season presented higher percentages of intact membrane (IM), non permeant (NP) membrane with high mitochondrial membrane potential (ΔΨm) and IM without PS translocation spermatozoa than those from the non-breeding (NB) season. Therefore, we can conclude that there were less spermatozoa showing apoptotic-like features in the seminal doses from the B than the NB season, although these differences did not affect field fertility. Only the percentage of intact membrane, non-capacitated (IM-NC) spermatozoa showed a significant correlation with in vivo fertility (P = 0.005) and fecundity (P = 0.007) values obtained after cervical AI when all data were evaluated. When the data were sorted by season and distance to the farms where AI was performed, the correlation between the percentage of IM-NC spermatozoa and reproductive parameters increased in the NB season and progressively with remoteness from the farms. Some other sperm parameters, like NP with high ΔΨm, IM sperm without active caspases and DNA-intact spermatozoa, also showed significant correlations with the reproductive parameters in the sorted data. Moreover, the increment in both the percentage of IM-NC and DNA-intact spermatozoa would increase the probability of obtaining a fertility higher than the mean (&gt;52%), as revealed by a multiple logistic regression analysis. In conclusion, we have identified two seminal markers—the percentage of intact membrane, non-capacitated spermatozoa, and DNA intact spermatozoa—which could be used as a test to discard males in AI programs, which is highly important from an economic point of view and can contribute to achieving satisfactory fertility rates.

Short Communication: Effect of cryopreservation on ultrastructure and mitochondrial function of albino Pangasius catfish spermatozoa

Hasanah U, Binawanto, Alimuddin A, Boediono A, Kursini E. 2020. Short Communication: Effect of cryopreservation on ultrastructure and mitochondrial function of albino Pangasius catfish spermatozoa. Biodiversitas 21: 4524-4528. Cryopreservation techniques have been carried out on many endangered species and animals with unique characteristics. Successful cryopreservation techniques vary between species depending on various factors. The study used cryopreserved spermatozoa of the albino Pangasius catfish as samples. The cryopreserved spermatozoa were analyzed by its ultrastructure, functional mitochondria, and viability. The cryopreservation was performed using a combination of 10% methanol, skim milk, and fish ringer extender. A deep freezer is used for cryopreservation at -80 °C with a storage period of 14 days. Observations were made on fresh spermatozoa, post-equilibration spermatozoa, and frozen-thaw spermatozoa. This study found there were differences in ultrastructure and morphology in the three treatments. Fresh spermatozoa and post-equilibration spermatozoa appeared intact membrane, mitochondrial, and flagellar structures. In contrast, in frozen-thaw spermatozoa, there was damage to the cell membrane. The study showed different percentages of yields on functional mitochondria of fresh spermatozoa (98 ± 2%), spermatozoa post-equilibration (57 ± 7%), frozen-thaw spermatozoa (42 ± 3.21%). Cell viability showed that there were differences in viability of fresh spermatozoa and frozen-thaw (p <0.05), the results of fresh spermatozoa (92 ± 0.57%) spermatozoa post-equilibration (80 ± 3.51%), frozen-thaw spermatozoa (61 ± 2.30%).The study concluded that the spermatozoa cryopreservation affects the ultrastructure, mitochondrial function, and viability in albino Pangasius catfish spermatozoa.

Major histocompatibility complex class 1 (MHC1) loss among patients with glioblastoma (GBM).

e14523 Background: Immune evasion represents a hallmark behavior of cancer and may occur through many mechanisms. Among these, intact tumor antigen presentation at the cell surface is a fundamental prerequisite to achieving successful adaptive immunotherapy. The loss of MHC1 expression due to molecular events, including mutation, deletion, or epigenetic silencing of B2M is commonly acquired during immunotherapy. On the other hand, molecular events affecting antigen presentation machinery may be present prior to immunotherapy administration. Among these, TP53 mutations causing loss of ERAP1 and TAP1 expression compromise transport of MHC1 molecules from the endoplasmic reticulum to the cell surface and mutant peptide integration into the HLA context resulting in absent antigen presentation. Thus far, clinical trials of PD-1/L1 agents have failed to demonstrate a benefit for GBM patients and responses are seen only among a minority. Hence, we set out to assess the integrity of MHC1 expression by using immunohistochemistry. Methods: Immunohistochemical (IHC) stains for HLA, B and C were developed and validated with internal controls. Staining intensity and location (membrane-bound or cytoplasmic) was evaluated semi-quantitatively. The first 10 consecutive patients with GBM who were referred for neoepitope vaccine were evaluated. Results: Absent staining was seen among 6/10, negligible, or faint staining was present in 2, and only 2 tumors demonstrated intact membrane-bound expression. Conclusions: In addition to low tumor mutation burden and an immunosuppressive tumor microenvironment, MHC1 loss is a frequent event among patients with GBM, and may be a dominant cause of immunotherapy failure for as many as 80% of patients. Thus, development of strategies to reverse this loss may be an essential component of successful adaptive immunotherapy for this disease. These data suggest that routine assessment of MHC1 should become a component of eligibility checking for GBM patients being considered for an MHC-restricted approaches. [Table: see text]