Normal Morphology Research Articles

Generation of a human embryonic stem cell line (SMUDHe010-A-1A) carrying Brainbow cassette in the AAVS1 gene by CRISPR/Cas9-mediated homologous recombination

Tracking single-cell lineages and their phenotypes longitudinally would help us better study skin development. Brainbow multicolor labeling approach is a genetic cell-labeling technique that tracks individual cells or analyzes cell lineages during development. Hence, we generated a stable Cre-inducible rainbow reporter human embryonic stem cell line (Named SMUDHe010-A-1A) by inserting the fluorescent protein gene (nGFP, YFP, RFP and CFP) at AAVS1 safe harbor locus using CRISPR/Cas9 technology. We verified that SMUDHe010-A-1A expressed the pluripotency markers and showed normal stem cell morphology. Furthermore, SMUDHe010-A-1A preserves normal karyotype and the ability to differentiate toward three germ layers. So the SMUDHe010-A-1A is effective for identifying and track cell populations.

Generation of a TBX20 homozygous knockout stem cell line (WAe009-A-1E) by episomal vector-based CRISPR/Cas9 system

The T-box family transcription factor gene TBX20 plays a crucial role in cardiac development and function. TBX20 mutations are associated with congenital heart disease, dilated cardiomyopathy, arrhythmias, and heart failure. To further study the role of TBX20 in human heart, here we generated a homozygous TBX20 knockout (TBX20-KO) human embryonic stem cell line using the CRISPR/Cas9 system. This TBX20-KO cell line maintains normal morphology, pluripotency, and karyotype, making it a valuable tool for investigating TBX20′s role in cardiac biology.

Generation of Leber congenital amaurosis, type 12 patient-specific induced pluripotent stem cell line (LVPEIi006-A), harboring a homozygous mutation in RD3

Leber congenital amaurosis (LCA) is a congenital, early onset, autosomal recessive inherited retinal disease (IRD). This report describes an LCA12 patient-specific iPSC line (LVPEIi006-A), generated by the reprogramming of dermal fibroblasts using integration-free episomal plasmids.This disease-specific iPSC model carries a homozygous point mutation in RD3, within the donor splice site at the end of exon 2 (c.296 + 1G > A). The stable line at passage 15 has displayed a normal colony morphology, expressed multiple stemness and pluripotency markers, lost all transgenes, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Enhancing growth, liver health, and bile acid metabolism of tilapia (Oreochromis niloticus) through combined cholesterol and bile acid supplementation in plant-based diets

The present study aimed to compare the nutritional effects of cholesterol, bile acids, and combination of cholesterol with bile acids in plant-based diets on juvenile genetically improved farmed tilapia (GIFT; Oreochromis niloticus). The isonitrogenous (321 g/kg crude protein) and isolipidic (76 g/kg crude fat) diets (Con diet) were based on plant protein sources, which included corn gluten meal, soybean meal, cottonseed meal and rapeseed meal. The Con diet was supplemented with 12 g/kg cholesterol (CHO diet), 0.2 g/kg bile acids (BAs diet), a combination of 12 g/kg cholesterol and 0.2 g/kg bile acids (CHO–BAs diet), respectively. Each diet was fed to three tanks in an indoor recirculating aquaculture system for 9 weeks. Results showed that compared to the Con group, fish had a higher weight gain rate, hepatosomatic index, and a lower feed conversion ratio in the CHO–BAs group. The highest levels of whole-fish fat and ash were found in the Con group. Serum parameters, including activities of alanine aminotransferase (ALT) and aspartate transaminase (AST), along with levels of glucose (GLU) and triglyceride (TG) except for total cholesterol (TCHO), were lower in the CHO, BAs, and CHO–BAs groups than those in the Con group (P < 0.001). Histological examination revealed that fish in the Con group exhibited severe hepatocyte vacuolization and diminished hepatocyte proliferation. Gene expression analysis indicated that the transcriptional levels of bile acid metabolism-related genes (including fxr, fgf19, bsep) were up-regulated in the CHO–BAs group (P < 0.05), whereas cholesterol metabolism-related genes (acly and hmgcr) were down-regulated in both CHO and CHO–BAs groups (P < 0.001). Moreover, UPLC-MS/MS analysis revealed that the higher taurine-conjugated bile acids (T-BAs), followed by free bile acids (Free-BAs) and glycine (G-BAs) were determined in tilapia bile. Among these, taurochenodeoxycholic bile acid was the predominant bile acid. Dietary bile acids supplementation also increased the proportion of T-BAs (tauro β-muricholic acid and taurodehydrocholic acid) while decreasing Free-BAs in the fish bile. In conclusion, the incorporation of cholesterol with bile acids into plant-based diets can effectively reduce cholesterol uptake, suppress bile acids synthesis, enhance bile acids efflux, and promote hepatocyte proliferation, which is helpful for maintaining the normal liver morphology in tilapia, and thus improving its growth performance.

Consumption of Citric Acid by Bees Promotes the Gland Development and Enhances Royal Jelly Quality.

The glands of bees are responsible for generating and secreting various biologically active substances that significantly impact bee physiological health and adaptability. This study aimed to investigate the effects of adding citric acid (CA) to bee feed on gland development and royal jelly quality. By formulating feed with varying proportions of CA, evaluation was undertaken of pollen feeding by honeybees under laboratory conditions, along with the impact of CA on the development of major glands, to determine suitable addition proportions. Further optimization of the CA proportion involved feeding colonies and evaluating royal jelly production and quality. The results indicated that feed containing 0.75% CA significantly extended the lifespan of bees and increased their pollen consumption. Gland development in bees showed a positive correlation with CA addition within the range of 0.25% to 0.75%, especially at 0.50% and 0.75%, which notably accelerated the development of mandibular, hypopharyngeal, and cephalic salivary glands, with active proliferation and differentiation of glandular cells and maintenance of normal gland size and morphology. CA added to feed stimulated vigorous secretion of wax glands in worker bees, resulting in prolific wax construction. Colonies consuming feed containing 0.50% CA produced royal jelly with significantly reduced moisture and total sugar content and increased levels of 10-HDA, total phenolic acids, total proteins, and acidity. These findings demonstrate that CA consumption significantly prolongs bee lifespan, increases consumption, promotes gland development, and enhances royal jelly quality. This research provides theoretical guidance for beekeeping practices and feed development, contributing to the sustainable advancement of apiculture.

Effect of Conditioned Medium from Human Adipose-Derived Mesenchymal Stem Cells on Human Sperm Quality During Cryopreservation.

The cryopreservation procedure decreases sperm quality, causing certain changes at structural and molecular levels affecting fertilizing ability. We aimed to investigate the impacts of human adipose-derived mesenchymal stem cells (HAd-MSCs) conditioned medium (CM) on the protection of human sperm from cryoinjury. Thirty normal semen specimens were evaluated in this study. Each specimen was separated into six groups and enhanced with varying concentrations of human Ad-MSCs-CM (0, 10, 30, 50, 70, and 100%). Sperm motility, viability, morphology, apoptosis, mitochondrial potential, and lipid peroxidation, and DNA fragmentation were evaluated before freezing and after thawing. The results showed that the total motility was preserved in 10% human Ad-MSCs-CM group. Also, DNA fragmentation was significantly lower in 10% compared to 0% human Ad-MSCs-CM (63.62 ± 17.72% vs.76.46 ± 4.87%, respectively, P < 0.004). Human Ad-MSCs-CM in groups of 10, 30, 50, and 70% reduced lipid peroxidation. The normal sperm morphology rate, mitochondrial membrane potential, and apoptosis showed no significant differences across various groups. It seems that human Ad-MSCs-CM can protect the sperm parameters during the cryopreservation by decreasing cryoinjury.

Will Nigella sativa oil protect parotid glands of rats against cranium gamma irradiation? Histological and immunohistochemical evaluation

BackgroundRadiation plays an essential role in treating malignancies. Radiation exposure of salivary glands often results in permanent loss of their functions; therefore, their protection against radiation is crucial. Nigella sativa oil (NSO) is a useful antioxidant against free radicals. The purpose of this study was to investigate the radio-protective effect of NSO on oxidative injury of parotid glands of gamma-irradiated rats. Methods: Twenty-eight male albino rats were divided into four groups (n = 7): Group 1: Neither NSO nor radiation, Group 2: Rats received NSO 400 mg/kg, Group 3: Rats received 15 Gy cranium gamma irradiation & Group 4: Rats received gamma irradiation and NSO. Rats were sacrificed two weeks after the last NSO dose. Histological sections of parotid glands were stained with H&E, Masson’s trichrome and anti-TGF-β antibodies. Area percentage of Masson’s trichrome and TGF-β expression was morphometrically examined. Results: Parotid glands of control and NSO groups revealed normal morphology. Gamma-irradiated glands showed loss of normal acinar architecture and slight acinar shrinkage. NSO treatment of gamma-irradiated glands preserved acinar outline and architecture. Masson’s trichrome stained samples revealed trace amounts of collagen fibers in control and NSO groups, and excessive amounts of collagen fibers in gamma-irradiated group, in addition to few collagen fibers for gamma-irradiated glands treated with NSO. Additionally, control and NSO groups showed negative TGF-β expression. Gamma-irradiated group showed high TGF-β expression, while NSO treated gamma-irradiated group showed moderate TGF-β expression. Conclusions: Gamma-irradiation adversely affected parotid glands, and in contrast, NSO seemed to positively counteract this adverse effect.

Mycobacterial FtsEX-RipC interaction is required for normal growth and cell morphology in rifampicin and low ionic strength conditions.

Mycobacterial cell growth and division are coordinated with regulated peptidoglycan hydrolysis. Understanding cell wall gene complexes that govern normal cell division and elongation will aid in the development of tools to disarm the ability of mycobacteria to survive immune-like and antibiotic stresses. We combined genetic analyses and scanning electron microscopy to analyze morphological changes of mycobacterial FtsEX and RipC mutants in stressful conditions. We demonstrate that FtsE, FtsX, FtsEX, and RipC are conditionally required for the survival of Mycobacterium smegmatis during rifampicin treatment and in low-salt conditions. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. We also show that the FtsEX-RipC interaction is essential for the survival of M. smegmatis in rifampicin. Our results suggest that FtsE, FtsX, and RipC are required for normal cell wall regulation and ultimately for survival in stressful conditions.

Living in a dynamic environment: The effects of multi-ways temperature variation on embryo and newborn juveniles of a shallow-water octopus (Amphioctopus fangsiao)

Shallow waters are characterized by fluctuating environmental conditions, modulating marine life cycles and biological phenomena. Multiple variations in water temperature could affect eggs and embryos during spawning events of many marine invertebrate species, yet most of the findings on embryonic development in invertebrates come from experiments based on the constant temperature. In this study, to examine the effects of temperature variation on octopus embryos, Amphioctopus fangsiao, a common shallow-water octopus along the coast of China, was exposed to the constant temperature (18 °C, in situ temperature of the seawater in Lianyungang), ramping temperatures (from 18 to 24 °C), diel oscillating temperatures (18 °C and 20 °C for 12 h each day), and acute increasing temperatures (the temperature increased sharply from 18 °C to 24 °C at embryonic development stage XIX) for 47 days (from embryogenesis to settlement). The results demonstrated that the temperature variations accelerated the development time of A. fangsiao embryos. Temperature fluctuations could cause embryonic oxidative damage and disorder of glycolipid metabolism, thereby affecting the growth performance of embryos and the survival rate of hatchings. Through transcriptome sequencing, the mechanistic adaption of the embryo to environmental temperature variations was revealed. The pathways involved in the TCA cycle, DNA replication and repair, protein synthesis, cell signaling, and nervous system damage repair were significantly enriched, indicating that the embryo could improve heat tolerance to thermal stress by regulating gene expression. Moreover, acute warming temperatures posed the most detrimental effects on A. fangsiao embryos, which could cause embryos to hatch prematurely from the vegetal pole, further reducing the survival of hatchings. Meanwhile, the diel oscillating temperature was observed to affect the normal morphology of the embryo, resulting in embryo deformities. Thus, the constant temperature is critical for balanced growth and defense status in octopuses by maintaining metabolism homeostasis. For the first time, this study evaluates the effects of multiple temperature fluctuations on embryos of A. fangsiao, providing new insights into the physiological changes and molecular responses of cephalopod embryos following dynamic temperature stress.

(212) The Questionable Clinical Utility of Sperm Morphology: Rate of Isolated Teratospermia in a Population of Fertile Men

Abstract Introduction Over the last several decades, sperm morphology has represented a standard component of semen analysis. However, its clinical utility is not well-defined, despite continual efforts by the WHO to address critiques of semen morphology criteria through increasingly strict guidelines. Issues with intra- and inter-laboratory technical variability, in addition to the subjective nature of the morphology assessment, have been thoroughly discussed in existing literature. Furthermore, current literature has highlighted the low predictive value of sperm morphology abnormalities in assisted fertility treatment (ART) and natural pregnancy outcomes. As of 2020, the American Society for Reproductive Medicine (ASRM) guidelines have noted that morphology alone is not highly predictive or diagnostic of infertility. However, past survey studies have demonstrated the pervasive belief among physicians of the value of sperm morphology, potentially leading to counseling patients towards more expensive ART methods. These concerns call into question the emphasis placed on sperm morphology assessment and its value in the clinical setting. Objective To better understand the impact of sperm morphology on fertility, we assessed sperm morphology in a population of known fertile men. Methods Healthy men over 18 years of age were recruited to provide one semen sample prior to vasectomy from March 2020 through November 2022. Inclusion criteria for the study was as follows: (1) patients were required to have biological children under the age of 5 years old, with no history of difficulty achieving pregnancy, and (2) had undergone no prior fertility procedures. Sperm morphology was analyzed using Papanicolaou stain with Kruger strict criteria by two technicians in an Andrology specialty laboratory per the WHO 5th edition manual. Results There were 83 men who participated in the study, 68 of whom (81.9%) had a complete sperm morphology assessment available. On average, participants were 37 years of age and had a mean BMI of 28 (22.8, 33.2). Of those with a smoking history available (n=61), 38 (62.3%) were non-smokers, 7 (11.5%) were former smokers, and 16 (26.2%) were current smokers. Patients had an average of 3 children with a mean youngest child age of 11.5 months old. There were 38 patients (55.9%) with 3% or lower normal sperm morphology, including two patients who had 0% normal morphology (Table 1). The most common morphologic abnormalities were head shape defects (n=59, 84.3%), followed by coiled tails (n=14, 20.3). Men 3% or lower normal sperm morphology were four times as likely to have both head and tail abnormalities than men with 4% or greater normal sperm morphology (n=8 vs n=2, respectively); half of these men also had lower than normal motility, whereas none of the men with 4% or greater normal sperm morphology had lower than normal motility. Conclusions More than half of fertile male patients (65.6%) had lower than normal sperm morphology in our study. The results of our study further question the clinical relevance of sperm morphology on fertility outcomes and if the current approach in assessing morphology is too strict. Disclosure No.

(203) Impact of Smoking on Sperm Chromatin Integrity

Abstract Introduction Semen analysis is an essential component of infertility evaluation, but it may still fail to detect subtle sperm defects present in patients with male factor infertility. Although estimates vary, approximately 15% of patients with male factor infertility have normal spermiograms It is necessary to identify diagnostic measures for male infertility that are easy to perform, inexpensive, and provide an accurate diagnosis. Tests that assess sperm quality should not only identify the ability of spermatozoa to reach the oocyte but also their ability to fertilize the oocyte and activate embryo growth. The estimation of sperm DNA damage fills the above need in the diagnosis of male infertility Abnormalities in the male genome characterized by damaged sperm DNA may be indicative for male subfertility regardless of routine semen parameters, and these parameters do not reveal sperm DNA defects Men with normal semen analysis may also have a high degree of DNA fragmentation, which can be a major cause of undiagnosed/unexplained infertility. Sperm DNA fragmentation may result from aberrant chromatin packaging during spermatogenesis, defective apoptosis before ejaculation or excessive production of reactive oxygen species (ROS) in the ejaculate. Objective To study the impact of cigarette smoking on sperm chromatin integrity in infertile patients. Methods The present study included eighty infertile male patients recruited from the Andrology Outpatient Clinic at Al-Azhar University Hospitals The patients were divided into two groups: - Group I: forty nonsmoker infertile males. (Control Group). Group II: forty cigarette smoker infertile males. (Study Group) Results The current study revealed that smoking increases the abnormality of sperm chromatin integrity in sperms of smokers. This is evident by the positive correlation of increased head stainability of sperms in smokers compared to nonsmokers. The value of partially stained sperm heads using aniline blue is calculated from the total staining score formula, this score was found to increase significantly among smokers in comparison to nonsmokers. In the present study, the increased severity of smoking (smoking index), was not correlated with the degree of sperm head stainability. This mean that, smoking insult on sperm chromatin integrity is crucial regardless the duration or the amount of the consumed cigarettes Conclusions There is a significant increase of sperm chromatin damage in smoker infertile patients than nonsmoker infertile patients and these damages may affect the quality of the ejaculated spermatozoa and decrease their fertility potential. Not only semen parameters but also sperm chromatin condensation is affected significantly by smoking. Aniline blue staining is an effective and inexpensive tool for assessment of the fertilization potentiality of infertile patients. A significant correlation was found between the percentage of spermatozoa with normal sperm chromatin condensation and normal morphology. Cigarette smoking may have a negative impact on sperm quality and DNA integrity. Therefore, it would be advisable for men to abstain from smoking to avoid decreased fertility. Disclosure No.

Generation of a ISL1 homozygous knockout stem cell line (WAe009-A-1G) by episomal vector-based CRISPR/Cas9 system

The ISL LIM homeobox 1 (ISL1) gene belongs to the LIM/homeodomain transcription factor family and plays a pivotal role in conveying multipotent and proliferative properties of cardiac precursor cells. Mutations in ISL1 are linked to congenital heart disease. To further explore ISL1′s role in the human heart, we have created a homozygous ISL1 knockout (ISL1-KO) human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this ISL1-KO cell line retains normal morphology, pluripotency, and karyotype. This resource serves as a valuable tool for investigating ISL1′s function in cardiomyocyte differentiation.

108 Discriminating highly resilient spermatozoa during long-term chilled storage

Abstract Artificial insemination (AI) is the most important assisted reproductive technique devised to enhance the genetic potential of animals by enabling widespread utilization of elite males. In the swine industry, AI with extended semen is highly effective, with several extenders developed for long-term storage. Regardless, the gradual decline of sperm quality during storage remains a major concern for rapid genetic dissemination. We hypothesized that existing sire-to-sire variations could reflect on sperm survival during prolonged storage. Therefore, this study examined boar sperm motility and morphology profiles to determine potential disparities during long-term storage within a commercial extender. Freshly collected Duroc boar semen samples (n = 58 boars) were extended at a commercial boar stud (Prestage Farms, MS), and sample doses (n = 29) were appropriately transported to the laboratory for experiments. Semen samples were aliquoted in tubes (filled to the top) and immediately stored in an incubator set at 17°C for up to 10 d post-collection. Daily, samples were assessed for total motility (TM), progressive motility (PM), velocities [average path velocity (VAP), curvilinear velocity (VCL), and straight-line velocity (VSL)], and normal morphology (NM) using the computer-assisted sperm analyzer (CASA, CEROS II). The deviations of TM means were used for survival discrimination between so-called “good” and “bad” semen. Data were log-transformed and analyzed (repeated ANOVA and Bonferroni adjustment) to determine the effect of time during storage. Results are expressed as mean ± SEM and P &amp;lt; 0.05 indicates significant differences. As expected, all data demonstrated a consistent and significant decline over time (P &amp;lt; 0.05), from day-0 to day-10 (TM: 78.2 ± 1.0% to 42.8 ± 4.3%; PM: 46.1 ± 1.8% to 16.2 ±2.3%, and NM: 85.1 ± 1.0% to 76.1 ± 1.9%). Notably, velocity parameters (VAP, VSL, and VCL) remained relatively stable during the storage period, while the distal droplets contributed the most to morphological defects (54.1±1.5% vs. 28.3±1.5%, 18.3±0.5%, and 2.4±0.2% for the proximal droplets, bent and coiled tailed, respectively; P&amp;lt; 0.05). Survival discrimination indicated the highest deviations from the overall averages on day-7.Consequently, good (n = 8) and bad (n = 7) semen samples had significantly different (P &amp;lt; 0.05) in TM (80.2 ± 1.4% and 21.7 ± 4.6%, respectively), PM (39.7 ± 2.7% and 4.7 ± 1.7%, respectively), and NM (87.9 ± 1.9 and 66.4 ± 2.6%, respectively).Similarly, the velocity parameters were significantly different (P &amp;lt; 0.05) between both sample groups, including VAP (74.9 ± 2.6 µm/s vs. 40.4 ± 8.7 µm/s), VCL (135.2 ± 6.0µm/s vs. 81.9 ± 17.3 µm/s), and VSL (59.0 ± 1.8 µm/s vs. 31.3 ± 6.8 µm/s). These findings indicate that specific subsets of boar semen may be well-suited for survival during long-term chilled preservation. The development of tools for early discrimination of semen doses, immediately following collection, could significantly enhance their management and facilitate appropriate shipments to remote areas or during adverse weather conditions. Ongoing studies are dedicated to unraveling the cellular mechanisms underlying the resilience of such spermatozoa. - Research funded by USDA-ARS, grant#58-6066-3-038.

Protective effect of co-enzyme Q10 on testicular tissue and sperm parameters in adult male rats treated with Sunset Yellow FCF

Objective: To determine the protective effect of co-enzyme Q10 (CoQ10) on testicular tissue and sperm parameters in male rats treated with Sunset Yellow FCF. Methods: Sixty male Sprague-Dawley rats were randomly divided into 6 groups of the control, CoQ10 (10 mg/kg/day), low dose of Sunset Yellow (2.5 mg/kg), high dose of Sunset Yellow (70 mg/kg), low dose of Sunset Yellow (2.5 mg/kg) plus CoQ10, and high dose of Sunset Yellow (70 mg/kg) plus CoQ10. The drugs were administered via daily oral gavages for 6 weeks. At the end of the experiment, sperm analysis, stereological and histological assessments of the testis were carried out. Results: The normal morphology (by 41.1%) and progressive spermatozoa (by 74.8%), testicle volume (by 33.4%), lumen volume (by 38.3%), interstitial tissue volume (by 44.7%), seminiferous tubule volume (by 40.7%), and number of spermatogonia (by 53.9%) and Leydig cells (by 70.7%) reduced in the rats that received high doses of Sunset Yellow in comparison to the control group. Nonetheless, all these alterations were recovered by CoQ10 treatment in the CoQ10 plus high dose of Sunset Yellow group. Furthermore, low doses of Sunset Yellow did not affect different parameters of the testis and sperm. Conclusions: CoQ10 could, to some extent, prevent structural changes of the testis induced by the high dose of Sunset Yellow.

Spem2, a novel testis-enriched gene, is required for spermiogenesis and fertilization in mice

Spermiogenesis is considered to be crucial for the production of haploid spermatozoa with normal morphology, structure and function, but the mechanisms underlying this process remain largely unclear. Here, we demonstrate that SPEM family member 2 (Spem2), as a novel testis-enriched gene, is essential for spermiogenesis and male fertility. Spem2 is predominantly expressed in the haploid male germ cells and is highly conserved across mammals. Mice deficient for Spem2 develop male infertility associated with spermiogenesis impairment. Specifically, the insufficient sperm individualization, failure of excess cytoplasm shedding, and defects in acrosome formation are evident in Spem2-null sperm. Sperm counts and motility are also significantly reduced compared to controls. In vivo fertilization assays have shown that Spem2-null sperm are unable to fertilize oocytes, possibly due to their impaired ability to migrate from the uterus into the oviduct. However, the infertility of Spem2−/− males cannot be rescued by in vitro fertilization, suggesting that defective sperm–egg interaction may also be a contributing factor. Furthermore, SPEM2 is detected to interact with ZPBP, PRSS21, PRSS54, PRSS55, ADAM2 and ADAM3 and is also required for their processing and maturation in epididymal sperm. Our findings establish SPEM2 as an essential regulator of spermiogenesis and fertilization in mice, possibly in mammals including humans. Understanding the molecular role of SPEM2 could provide new insights into future therapeutic treatment of human male infertility and development of non-hormonal male contraceptives.

Preconditioning exercise reduces brain damage of ischemic stroke in rats via PI3K-AKT pathway by bioinformatic analysis.

Ischemic stroke is one of the most vital causes of high neurological morbidity and mortality in the world. Preconditioning exercise is considered as the primary prevention of stroke to resistance to subsequent injury. We tried to research the underlying biological mechanisms of this exercise. Forty-two SD rats were randomly divided into three groups: middle cerebral artery occlusion (MCAO) group, exercise group with MCAO (EX + MCAO) group, and sham group, with 14 rats in each group. The EX + MCAO group underwent exercise preconditioning for 3weeks before occlusion, and the other two groups were fed and exercised normally. After 3weeks, MCAO model was made by thread plug method in the EX + MCAO group and MCAO group. After successful modeling, the Longa scale was used to evaluate the neurological impairment of rats at day 0, day 1, and day 2. The rats in each group were killed on the third day after modeling. TTC staining measured the infarct volume of each group. The morphology and apoptosis of cortical cells were observed by HE and Tunel staining. Three rats in each group underwent high-throughput sequencing. Bioinformatic analysis was used to find the deferentially expressed genes (DEGs) and predict the transcription factor binding sites (TFBS) of the next-generation sequencing results. Gene enrichment (GSEA) was used to analyze potential functional genes and their corresponding signaling pathways. The Longa scale showed EX + MCAO group had the neurological function better than the modeling group (P < 0.001). TTC staining showed that the infarct size of EX + MCAO group was less than MCAO group (P < 0.05). HE and Tunel staining showed that the cells in the EX + MCAO group and the sham group had normal morphology and fewer apoptotic cells than MCAO group. A new gene named 7994 was discovered and TFBS of this gene was predicted, which could interact with key genes such as Foxd3, Foxa2, NR4A2, SP1, CEBPA, and SOX10. GSEA showed that EX + MCAO group could promote and regulate angiogenesis and apoptosis through PI3K-AKT pathway. Preconditioning exercise could improve nerve function and reduce infarct size in rats. The underlying mechanism is to regulate the PI3K-AKT pathway through several key genes, promote cerebral angiogenesis, and reduce apoptosis.

Agrobacterium Transformation of Tea Plants (Camellia sinensis (L.) KUNTZE): A Small Experiment with Great Prospects.

Tea has historically been one of the most popular beverages, and it is currently an economically significant crop cultivated in over 50 countries. The Northwestern Caucasus is one of the northernmost regions for industrial tea cultivation worldwide. The domestication of the tea plant in this region took approximately 150 years, during which plantations spreading from the Ozurgeti region in northern Georgia to the southern city of Maykop in Russia. Consequently, tea plantations in the Northern Caucasus can serve as a source of unique genotypes with exceptional cold tolerance. Tea plants are known to be recalcitrant to Agrobacterium-mediated transfection. Research into optimal transfection and regeneration methodologies, as well as the identification of tea varieties with enhanced transformation efficiency, is an advanced strategy for improving tea plant culture. The aim of this study was to search for the optimal Agrobacterium tumefaciens-mediated transfection protocol for the Kolkhida tea variety. As a result of optimizing the transfection medium with potassium phosphate buffer at the stages of pre-inoculation, inoculation and co-cultivation, the restoration of normal morphology and improvement in the attachment of Agrobacterium cells to the surface of tea explants were observed by scanning electron microscopy. And an effective method of high-efficiency Agrobacteria tumefaciens-mediated transfection of the best local tea cultivar, Kolkhida, was demonstrated for the first time.

Beta cell specific cannabinoid 1 receptor deletion counteracts progression to hyperglycemia in non-obese diabetic mice

ObjectiveType 1 diabetes (T1D) occurs because of islet infiltration by autoreactive immune cells leading to destruction of beta cells and it is becoming evident that beta cell dysfunction partakes in this process. We previously reported that genetic deletion and pharmacological antagonism of the cannabinoid 1 receptor (CB1) in mice improves insulin synthesis and secretion, upregulates glucose sensing machinery, favors beta cell survival by reducing apoptosis, and enhances beta cell proliferation. Moreover, beta cell specific deletion of CB1 protected mice fed a high fat high sugar diet against islet inflammation and beta cell dysfunction. Therefore, we hypothesized that it would mitigate the dysfunction of beta cells in the precipitating events leading to T1D. MethodsWe genetically deleted CB1 specifically from beta cells in non-obese diabetic (NOD; NOD RIP Cre+ Cnr1fl/fl) mice. We evaluated female NOD RIP Cre+ Cnr1fl/fl mice and their NOD RIP Cre−Cnr1fl/fl and NOD RIP Cre+ Cnr1Wt/Wt littermates for onset of hyperglycemia over 26 weeks. We also examined islet morphology, islet infiltration by immune cells and beta cell function and proliferation. ResultsBeta cell specific deletion of CB1 in NOD mice significantly reduced the incidence of hyperglycemia by preserving beta cell function and mass. Deletion also prevented beta cell apoptosis and aggressive insulitis in NOD RIP Cre+ Cnr1fl/fl mice compared to wild-type littermates. NOD RIP Cre+ Cnr1fl/fl islets maintained normal morphology with no evidence of beta cell dedifferentiation or appearance of extra islet beta cells, indicating that protection from autoimmunity is inherent to genetic deletion of beta cell CB1. Pancreatic lymph node Treg cells were significantly higher in NOD RIP Cre+ Cnr1fl/flvs NOD RIP Cre−Cnr1fl/fl. ConclusionsCollectively these data demonstrate how protection of beta cells from metabolic stress during the active phase of T1D can ameliorate destructive insulitis and provides evidence for CB1 as a potential pharmacologic target in T1D.

Sperm binding to oviduct epithelial spheroids varies among males and ejaculates but not among females in pigs

The elimination of ejaculates and males with low fertility despite good sperm motility and morphology is crucial to maintain high pregnancy rates after artificial insemination (AI) in farm animals. The ability of sperm to survive in the female tract is particularly crucial in pigs due to the large variation in the timing between AI and ovulation and the high number of oocytes to fertilise. The objective of this study was to characterise a new in vitro model of oviduct sperm reservoir using porcine oviduct epithelial spheroids (OES) and to assess the variability in sperm binding to OES among gilts, boars and their ejaculates. Isthmic mucosa fragments were collected from gilt oviducts at a slaughterhouse, and after 48 h of culture, the OES that had spontaneously formed were sorted according to their vesicle shape and size (150–200 μm in diameter) for characterisation and sperm binding assays. The OES contained viable, cytokeratin-positive and vimentin-negative cells, of which 36.4 ± 2.0% were multiciliated. The average proportion of multiciliated cells per OES did not change among culture replicates. After co-incubation with boar fresh semen, only sperm of normal morphology were found to bind, by their head, to cilia of OES. The density of sperm bound to the OES surface increased linearly with sperm concentration. The bound sperm density on OES was used to assess the binding capacity of fresh ejaculates collected from Pietrain boars. For a given ejaculate, the bound sperm density did not vary among pools of OES female donors. The analysis of five successive ejaculates from nine boars indicated significant differences in bound sperm densities on the OES among individual boars and their ejaculates (P < 0.01). There was no correlation between the sperm bound density and sperm parameters measured by computer-assisted sperm analysis or the initial dilution of the ejaculate. In conclusion, the OES characterised in this study offered physiological conditions to study sperm binding to the isthmic reservoir and evidenced that sperm from different ejaculates and different boars vary in their ability to bind to these oviduct spheroids despite homogeneous motility and morphology.

Relationship between dietary glycemic index and glycemic load and sperm-quality parameters in Iranian men: a cross-sectional study

BackgroundInfertility is a major clinical problem with psychological, financial and medical costs. Male infertility has recently been linked to 50% of childless couples. It is worth mentioning that diet and its components can be manipulated and applied in counseling meetings of infertile men as a modifiable factor. The goal of this study was to determine the correlation of dietary glycemic index (GI) and glycemic load (GL) with sperm-quality parameters in Iranian men.Materials and methodsIn this cross-sectional study which was carried from Aug to Nov 2023, after excluding smokers, 322 men who were attending the IVF clinic of Ayatollah Mousavi Hospital for seminal analysis were enrolled. Dietary intake was completed by an expert dietitian through face-to-face interviews with a validated 168-item food frequency questionnaire (FFQ). In the present study, sperm-quality parameters, including motility, concentration, normal morphology, vitality, DNA fragmentation index (DFI), and chromatin maturation of sperm, were analyzed. The relationship between dietary GI and CL and these parameters was assessed.ResultsAdjusting for the baseline variables, dietary GI and GL showed positive and negative associations with sperm progressive motility, respectively (p = 0.01 and p < 0.001). Higher dietary GI was associated with lower (p = 0.03); however, a higher dietary GL was associated with higher immotile sperm (p < 0.001). A higher dietary GI was associated with a 77% higher sperm count (p = 0.003). In contrast, higher dietary GL was associated with a lower count (p < 0.001). Higher dietary GI and GL were associated with higher and lower percentages of sperm with normal morphology by 70% and 40%, respectively (p < 0.001 in both). A higher dietary GL was associated with an increase in sperm vitality and DFI of 33% and 53%, respectively (p < 0.001). Higher dietary GI showed a significant negative effect on sperm DFI (p = 0.009). Dietary GI and GL showed significant positive and negative effects on SCMA, respectively (p = 0.002 and p < 0.001).ConclusionA diet with a higher GI showed beneficial effects on more parameters of sperm; however, higher dietary GL showed deleterious effects, except sperm vitality and SCMA. More studies are needed to obtain a concise result.