Most studies examining survival of neonates with congenital diaphragmatic hernia (CDH) are in high-income countries. We aimed to describe the management, survival to hospital discharge rate, and factors associated with survival of neonates with unilateral CDH in a middle-income country.We retrospectively reviewed the medical notes of neonates with unilateral CDH admitted to a pediatric intensive care unit (PICU) in a tertiary referral center over a 15-year period, from 2003–2017. We described the newborns’ respiratory care pathways and then compared baseline demographic, hemodynamic, and respiratory indicators between survivors and non-survivors. The primary outcome measure was survival to hospital discharge.p > 0.05), from 56.0% to 69.5% before and after, respectively, a service reorganization. For those neonates who could be stabilized and operated on, 90.9% (80/88) survived to hospital discharge. The commonest post-operative complication was infection, occurring in 43.3%. The median survivor length of stay was 32.5 (interquartile range 18.8–58.0) days. Multiple logistic regression modelling showed vaginal delivery , Apgar score p = 0.028), and fraction of inspired oxygen (FiO2) < 50% at 24 h  were significantly associated with improved survival to hospital discharge.Altogether, 120 neonates were included with 43.3% (52/120) diagnosed antenatally. Stabilization occurred in 38.3% (46/120) with conventional ventilation, 13.3% (16/120) with high-frequency intermittent positive-pressure ventilation, and 22.5% (27/120) with high frequency oscillatory ventilation. Surgical repair was possible in 75.0% (90/120). The overall 30-day survival was 70.8% (85/120) and survival to hospital discharge was 66.7% (80/120). Survival to hospital discharge tended to improve over time (2 < 50% at 24 h increased the likelihood of survival to hospital discharge.We report a survival to hospital discharge rate of 66.7%. Survival tended to improve over time, reflecting a greater critical volume of cases and multi-disciplinary care with early involvement of the respiratory team resulting in improved transitioning from PICU. Vaginal delivery, Apgar score  Congenital diaphragmatic hernia (CDH) is a severe diaphragmatic malformation that permits abdominal organ herniation into the chest cavity. It is rare, with population studies giving an estimated prevalence of 2.3 per 10,000 live births [Treatment of CDH is challenging, requiring the expertise of integrated multidisciplinary teams in well-resourced intensive care units. The aim is to gradually recruit the lungs and simultaneously minimize lung injury, which can be achieved using ‘gentle ventilation’ with permissive hypercapnia –4. High-Population-based studies from the 1970s to 1990s, which included elective termination and still-born neonates, consistently reported CDH mortality rates ranging from 61–66% , 9, 10. The CDH Study Group (CDHSG) initiated a registry, collecting multi-institutional data from many countries, to evaluate variations in treatment and outcome, with the ability to adjust for severity . The regOver the past two decades, studies from South and Southeast (SE) Asian countries, mostly involving relatively small numbers of neonates and with varying selection criteria, report survival rates ranging from 56–78% in India –18, 52% The objectives of this study were to describe the management and survival rates of neonates with CDH admitted to a non-ECMO pediatric intensive care unit (PICU) in a middle-income country in SE Asia over a 15-year period, and to analyze the factors associated with survival.We performed a retrospective cohort study including all liveborn neonates diagnosed with unilateral CDH from 2003–2017, admitted to the PICU, University Malaya Medical Center, Kuala Lumpur, Malaysia.-i ventilator . From 2015, a Fabian Ventilator  was added. The ventilation strategy followed an established protocol based on Wung et al. [2O, and pressure above PEEP of 15 to 20 cm, to obtain a peak inspiratory pressure (PIP) of 20 to 25 cm H2O for adequate chest/abdominal excursion. Supplemental oxygen was administered to maintain pre-ductal oxygen saturation (SpO2) > 90%. Permissive hypercapnia with a pH > 7.25 was considered acceptable. Neonates who did not respond were switched to high-frequency intermittent positive-pressure ventilation (HIPPV) using the same ventilator but with a respiratory rate of 100 breaths/min, pressure above PEEP of up to 20 cm H2O, and PEEP of 0 cm H2O [2O. In cases with persistent hypoxemia, labile oxygenation, or if the partial pressure of carbon dioxide (PaCO2) > 60 mm Hg, HFOV was used as the rescue mode [The respiratory care strategy for newborns diagnosed with CDH followed a standard protocol, which was modified over the 15-year period as new evidence and infant ventilators became available. All infants were intubated as soon as the CDH diagnosis was recognized and ventilated with conventional ventilation using a SERVOg et al. : respira0 cm H2O  but withcue mode .2 > 88% with minimal but adequate ventilator settings. Hyperventilation to induce alkalosis and hyperinflation of the hypoplastic lungs were avoided by reducing the ventilation rate and the pressure above PEEP. Inotropic support using dopamine, noradrenaline, adrenaline, and/or milrinone, and calcium infusions were used to maintain mean arterial blood pressure ≥ 40 cm H2O, which minimized right-to-left shunting across the ductus arteriosus. Echocardiography to monitor heart function, intravascular volume status, right and left ventricular functions and to diagnose coexisting cardiac defects was conducted as soon as the neonate’s condition permitted.In the presence of PPHN, iNO therapy at 20 ppm was initiated , 24. Mag2) < 50%, the patient was weaned off iNO and all inotropes. Post-operatively, patients were continued on conventional ventilation and when stabilized were extubated to non-invasive ventilation.Surgery was performed when the neonate’s general condition and hemodynamic parameters stabilized for at least 24 h. After transitioning to conventional ventilation and a fraction of inspired oxygen , surgical repair (yes/no), stabilization achieved prior to surgery (yes/no), defect size  as used by the CDHSG [All neonates with CDH were identified from PICU admission records, their case notes reviewed, and the following information was extracted: baseline demographic and perinatal characteristics . For continuous data, the Mann–Whitney U test was used. Univariate associations with survival were assessed. Adjusted differences in survival, based on pre-operative indicators, were estimated utilizing multiple logistic regression models. Criteria for entry and removal from this model were a p < 0.05 and p > 0.10, respectively. The final multivariate model chosen was one that best represented the data. Data were analyzed using SPSS Statistics for Windows, version 23.0 . P-values < 0.05 were considered statistically significant.A descriptive analysis was undertaken for all study participants. Categorical data are described using numbers and percentages. The Shapiro–Wilk test was used to assess normal distribution. If continuous data were normally distributed, they are described using mean and standard deviation (SD); if not normally distributed, median, and interquartile range (IQR) are used. Univariate associations between pre-operative respiratory, pre-operative hemodynamic, and surgical characteristics and survival to hospital discharge were assessed using the chi-squared test for categorical data (Fisher’s exact test if p < 0.001). None of the children who were referred to us antenatally proceeded with elective termination. Altogether 41.2% of neonates (48/117) had associated anomalies, but only 14.5% (17/117) were deemed significant cardiac or syndromic anomalies — as defined by the CDHSG [Altogether 120 neonates were diagnosed with CDH, with 59.2% males (71/120). See Table nates 48/7 had assFigure n = 90), five neonates died before 30 days-of-life . Of the five patients who died after 30 days, two died without leaving PICU, one of PPHN and one of tracheobronchomalacia; the remaining three died after discharge from PICU, one of aspiration pneumonia due to a dislodged feeding tube, one of tracheobronchomalacia, and one of complications post-surgery for an interrupted aortic arch. In total, 69.2% (83/120) were discharged alive from PICU.Surgical repair was carried out in 75.0% (90/120) patients, including two cases who did not achieve stabilization pre-operatively, at a median age of 7.0 (IQR 5.0–12.0) days. The remaining 25.0% (30/120) of cases died without operation. Of those who underwent surgical repair , and the survival to hospital discharge rate was 66.7% (80/120). Of all those neonates who were stabilized and operated on, 90.9% (80/88) survived until hospital discharge. For those discharged home, the median length of hospital stay was 32.5 (IQR 18.5–58.0) days.p = 0.184). Those with no complications had a significantly lower median length of hospital stay than those who had complications . Four infants required additional surgery within this admission: two required aortopexy for tracheobronchomalacia, one had repair of an interrupted aortic arch, and one had ligation of a large patent ductus arteriosus.Post-operative complications were present in 47.8% (43/90) neonates. The commonest complication was blood stream infection 20.0% (18/90), followed by ventilator associated pneumonia 12.2% (11/90), wound or surgical site infection 10% (9/90), pneumothorax 3.3% (3/90), small bowel ischemia 1.1% (1/90), and infection site unspecified 1.1% (1/90). Those who survived to hospital discharge tended to have less complications than those who did not  in period 2 from 56.0% (14/25) in period 1 (p > 0.05). The percentage of low-risk defects (size A/B) was similar across both time periods, 81.2% (13/16) and 83.3% (60/72) for periods 2 and 1, respectively, (p > 0.05) Furthermore, there were significant reductions in median length of hospital and PICU stays in period 2 compared with period 1. The median length of hospital PICU stay was 21 (IQR 11–36.3) days vs. 33 (IQR 20.5–53.3) days (p = 0.044) and the median length of hospital stay was 31  vs. 72 (IQR 39.5–133.5) days (p = 0.005), for period 2 compared with period 1, respectively.The outcomes were analyzed into two time periods: 2000–2007 (period 1) and 2007–2017 (period 2), before and after a service reorganization, respectively. The number of cases antenatally diagnosed was significantly higher in period 2 compared with period 1 at 48.4% (46/95) vs. 24% (6/25), respectively, p > 0.05). Factors significantly associated with non-survival were antenatal diagnosis, being inborn, 1- and 5-min Apgar scores < 7, Cesarean section (elective and emergency) as the mode of delivery, high-risk defect (C or D), presence of pulmonary hypertension or preoperative air leak, non-stabilization preoperatively, lower initial pO2, lower initial pH, higher initial pCO2, and at 24 h lower pre- and post-ductal SpO2 and FiO2p = 0.001).Table p = 0.001 or less). A similar pattern was shown for OI; however, values were only significant at 0 and 6 h (both p < 0.001).Figure p = 0.041), Apgar score p = 0.028), and FiO2 < 50% at 24 h  were significantly associated with survival to hospital discharge.The variables used to predict survival in the final logistic regression model are shown in Table 2 < 50% at 24 h were significantly associated with survival to hospital discharge.We described the management and survival of 120 liveborn neonates with unilateral CDH over a 15-year period, in our tertiary referral center PICU, in a middle-income country. Altogether, three-quarters of patients had surgical repair and two-thirds survived to hospital discharge. The median length of hospital stay for survivors was 32.5 days. Vaginal delivery, Apgar score p > 0.05) [p < 0.006) [p < 0.001 [The survival to hospital discharge rate in our center at 66.7% was lower than the 72% rate found in the CDHSG, the latter study having 6948 neonates spanning 12 years until mid- 2019; however, this difference was not statistically significant ( > 0.05) . Altoget < 0.001 . ConversThe survival to discharge rate tended to improve with time in our unit, reaching around 70% in the last ten years of analysis. This may be due to several factors following a service reorganization from 2007: the provision of a dedicated multi-disciplinary, antenatal counselling service; greater experience and organization of the team; improved transitioning from the higher-intensity PICU to the lower-intensity pediatric ward with early involvement of the respiratory team; and increasing recognition of our center as one that manages CDH, resulting in a greater patient volume over time. It has been recognized that a critical volume of at least six cases annually may partly explain better survival in some centers , particun = 24 for both) and may reflect differing antenatal detection rates, termination rates, associated birth anomalies, ease of access to tertiary care facilities, and clinical management. Another study from Singapore found that 46% of cases diagnosed antenatally underwent termination [This is the largest study of CDH patients that we have found in SE Asia. Other smaller SE Asian studies have shown survival of patients ranging from 52–79% , 19–22. A recently published Danish study spanning a similar period, of equal size to ours, based in a tertiary referral unit without ECMO, and consisting of a similar percentage of antenatally detected cases had a 78% one-year survival . Our stup = 0.08). Indeed, in this antenatally diagnosed subgroup, survival remained higher following vaginal delivery at 65.4% (17/27) compared with Cesarean section at 34.6% (9/25), but the significance was borderline (p = 0.052). By contrast, antenatally diagnosed cases in the CDHSG showed a tendency for Cesarean section to improve survival, though findings were not significant; the authors excluded those with major heart defects and highlighted potential bias as centers that favor Cesarean section may have better survival rates [We found that vaginal delivery was associated with a significantly increased chance of survival. In our center, CDH neonates with an antenatal diagnosis were permitted to deliver vaginally unless there were specific contraindications. Typically, the pregnant woman was induced at 38/39 weeks’ gestation, if spontaneous onset of labor had not yet occurred. Although the evidence supporting the best route is uncertain, a spontaneous vaginal delivery maximizes gestational maturity . Howeveral rates . HoweverUltimately neonatal survival will depend on the degree of hypoplasia of the lungs and the ability to recruit them with ventilation and without causing pulmonary injury. It is unsurprising that univariate risk factors for mortality include a lack of response to any of the graded ventilation strategies and indicators such as OSI at all time intervals up to 48 h after delivery. As an indicator of hypoxemic respiratory failure, OI has been shown to predict mortality in CDH . We show2 < 50% at 24 h, were significantly associated with survival. These indicators have been found to significantly affect survival in studies of larger cohorts, are easily measured at the bedside, and are used in CDH survival prediction tools [2 of < 50% at 24 h was chosen as a variable for analysis because it has been suggested that starting ventilation with this FiO2 value may be more beneficial than at 100% [2 < 50% in the first 24 h, while only 22.9% (16/70) of survivors had an FiO2 > 50% at this time.An immediate postnatal marker of respiratory function, Apgar score on tools . An FiO2 at 100% . Of all n = 62) had a 45% infection rate, with no associations with mortality but significant associations with prolonged stay [Almost half of neonates who had surgery had a post-operative complication; 38.0% had infection. These are vulnerable neonates with immunological immaturity, undergoing multiple invasive procedures. A prospective observational study of CDH neonates in a neonatal intensive care unit in France . We have measured short-term outcomes only with no assessment of long-term morbidities, which can be considerable , 12. Som2 < 50% at 24 h were significantly associated with survival to hospital discharge.In this middle-income country, neonates with unilateral CDH who reached PICU and were stabilized had an excellent chance of surviving to hospital discharge. Vaginal birth route, Apgar score


Mammalian spermatogenesis is a heat-vulnerable process that occurs at low temperatures, and elevated testicular temperatures cause male infertility. However, the current reliance on in vivo assays limits their potential to detail temperature dependence and destructive processes. Using ex vivo cultures of mouse testis explants at different controlled temperatures, we found that spermatogenesis failed at multiple steps, showing sharp temperature dependencies. At 38 °C (body core temperature), meiotic prophase I is damaged, showing increased DNA double-strand breaks (DSBs) and compromised DSB repair. Such damaged spermatocytes cause asynapsis between homologous chromosomes and are eliminated by apoptosis at the meiotic checkpoint. At 37 °C, some spermatocytes survive to the late pachytene stage, retaining high levels of unrepaired DSBs but do not complete meiosis with compromised crossover formation. These findings provide insight into the mechanisms and significance of heat vulnerability in mammalian spermatogenesis. High temperatures lead to DNA double-strand break accumulation and asynapsis that trigger the meiotic checkpoint and apoptosis of spermatocytes in ex vivo cultures, providing a rationale for the heat sensitivity of sperm production in mammals. In many mammalian species, the testes descend into the scrotum at a later stage after developing deep in the abdominal cavity2. Compared with the body core, the scrotal temperature is maintained to 2–6 °C lower by means of heat radiation from the skin surface and counter-current heat exchange between arterial and venal blood flows3. When testes experience temperature elevation, spermatogenesis is compromised, leading to infertility4.Mammalian spermatogenesis is a heat-sensitive process that occurs under low-temperature conditions5. Cryptorchidism, a pathological condition in which the testes do not descend completely but remain in the abdominal cavity or inguinal canal, causes spermatogenesis failure4. Varicocele, in which the heat exchange between the testicular artery and vein is affected, is also a significant cause of male infertility4. Testicular temperatures are increased in these conditions, whose surgical correction can improve spermatogenesis and fertility6. In these high-temperature situations, the stage and extent of spermatogenesis defects are variable7, and the precise temperature dependence and mechanisms underlying such defects remain largely unresolved.From a clinical perspective, heat vulnerability during spermatogenesis is a well-recognized problem related to a broad spectrum of risk factors underlying male infertility in humans8. Artificial cryptorchidism, that is, surgical translocation of the testis into the abdominal cavity, is a widely used assay to study the effect of high temperatures of the body core on spermatogenesis13. In some studies, mice were exposed to elevated air temperatures15.Various in vivo experiments have been used to study heat sensitivity in mammalian spermatogenesis. In a classic study using pigs, naturally occurring cryptorchid testes recovered spermatogenesis when cooled by circulating water, demonstrating that high temperatures cause spermatogenesis defects13. In severest cases, all germ cells except undifferentiated spermatogonia are lost within a couple of weeks after surgical translocation, indicating that the transition from undifferentiated-to-differentiating spermatogonia is blocked. Such severe outcomes occur reproducibly if surgery follows a rigorous method, with the testes wrapped by the associated fat pad and positioned deep in the abdomen13. However, less thorough surgeries result in inconsistent testicular position and variable defects wherein, in some cases, only spermatogonia and spermatocytes remain, while round spermatids may persist in others13. A similar variability occurs in human testes with varicocele7. Thus, heat may affect multiple steps of spermatogenesis, possibly at different temperatures. However, the limitations of artificial cryptorchidism hinder the detailed understanding of temperature sensitivity. Foremost, the testicular temperature cannot be reliably measured or controlled. Furthermore, the effect of extratesticular factors mediated by other organs, hormones17, or the nervous system cannot be excluded.Artificial cryptorchid testes exhibit variable defects19. However, such experiments cannot measure or control the actual testicular temperature or exclude extratesticular factors, either. Furthermore, it is unclear if the results of transient heating at non-physiologically high temperatures can be extrapolated to chronic exposure to temperatures around the body core.Transient heat shock, which provides consistent results in short time courses, has been used to elucidate the mechanisms of spermatogenesis heat impairment. Lower-body bathing of mice in hot water  elicits stress response pathways and acute loss of spermatocytes and spermatids20. This setting supports the entire process of spermatogenesis over several weeks, leading to the production of functional sperm, albeit less efficiently than that produced in vivo. Using this culture, we investigated the chronic effect of precisely controlled temperatures on mouse spermatogenesis under conditions free from extratesticular factors.Previously, we developed an ex vivo air-liquid interface culture of mouse testicular explants14. The measured average temperatures were 34.0 °C and 37.8 °C in the scrota and body core, respectively, establishing the reported low-temperature condition in the scrota4  mice were cultured in an air-liquid interface  expression was activated in these mice during meiotic prophase I under an acrosin promoter. Precisely, GFP expression starts in pachytene spermatocytes that are in stage IV seminiferous epithelium and continue through subsequent steps of meiosis and the formation of round and elongating spermatids22  , spermatogenesis did not reach the late pachytene stage. Grades 1–4 reflect the extent of spermatogenesis progression beyond this stage , SCP3 (spermatocytes24), KIT (differentiating spermatogonia26), GFRα1 (a fraction of undifferentiated spermatogonia responsible for stem cell activity29), and GATA4 (Sertoli cells30)  Fig. , and theer) Fig. . Based o20 , the explants showed Acr-GFP fluorescence at nearly full grade from 2 to 5 weeks, as reported previously20 Fig. . In most 20 Fig. . These flls Fig. . In contlls Fig. . Consist °C Fig. . In cont °C Fig. . These fTo determine the temperature dependence of spermatogenesis, we incubated the explants at various temperatures ranging from 30 to 40 °C Fig. . The var+ spermatocytes were the most advanced type of germ cells  crossover(s)43. Crossover forms chiasmata as homologues detach in diplotene.We further investigated the mechanisms by which meiotic prophase I failed. In the normal process Fig. , numerou44. In leptotene and zygotene, newly formed DSBs elicit γH2AX generation, a process mediated by ATM45. γH2AX then decreases as the DSBs are repaired. Following this first wave, the second wave of γH2AX generation also occurs at later stages in response to unsynapsed chromosomes, known as the meiotic silencing of unsynapsed chromatin (MSUC)46. In in vivo samples, as reported, intense γH2AX signals were detected in the nuclei of leptotene and zygotene spermatocytes, but the signals were greatly reduced in pachytene, except for the intense signal in the XY-body reflecting the MSUC44 C44 Fig. . These oC44 Fig. .In testes grown ex vivo at 34 °C, γH2AX staining was similar to that of the in vivo control Fig. . At 37 °42.To determine whether DSB repair was impaired at high temperatures, we examined the localization of RPA2, RAD51, and DMC147. In explants grown at 34 °C, the number of RPA2 foci was slightly smaller than in vivo samples. A larger decrease was observed at 37 °C, whereas comparable numbers of RPA2 to that in in vivo samples were observed at 38 °C , a criterion which we followed in this study  pachytene Fig. . We thenely Fig. . In explely Fig. . This obely Fig. . Given tely Fig. . Thus, cCaenorhabditis elegans, a transient heat shock  elicits dysregulated expression of transposons and an increase in RAD51 foci in spermatocytes57. Heat-induced transposon activation also occurs in other systems such as Arabidopsis58. Furthermore, in mice, mutants deficient in the piRNA pathway exhibit dysregulation of transposons  and apoptotic death of spermatocytes63.Finally, we examined the potential involvement of dysregulated transposon expression, considering the increase in DSBs in explants cultured at 37 °C and 38 °C Fig. . In Caen64. On day 2, therefore, the surviving spermatocytes are likely already impaired and about to die. However, we found that LINE1-ORF1 expression remained limited to a minor fraction of seminiferous tubules on day 2, contrary to the upcoming ubiquitous germ cell death , single-stranded DNA binding proteins , DSB repair machinery , or their recruitment  result in apoptotic cell death at pachytene75. Further, showing dysregulated transposon expression associated with increased DSBs, various mutations in the piRNA pathway cause apoptosis at the meiotic checkpoint61.Further, we investigated meiosis failure observed at 38 °C and 37 °C and found an increase in DSBs and asynapsis. We reason that the 20, although there is a slight increase in DSBs and a small reduction in crossovers. At 38 °C, however, much greater DSBs occur at leptotene and zygotene, at least in part, due to defective DSB repair machinery . Abundant DSBs cause severe asynapsis between homologous chromosomes, leading to aberrant pachytene spermatocytes. In these cells, unrepaired DSBs and asynapsis exceed the threshold, activating the meiotic checkpoint for apoptosis. At 37 °C, the damaged cells show a broad range of DSBs and asynapsis, from levels similar to 34 °C to those comparable to 38 °C, reflecting the variable defects in DSB repair foci assembly. Here, cells carrying DSBs and asynapsis above the threshold may result in aberrant pachytene and undergo apoptosis. While, cells whose DSBs and asynapsis are below the threshold complete the chromosome pairing and are licensed to proceed to late pachytene. However, these cells do not survive beyond meiotic divisions, with unrepaired DSBs and asynapsis at levels higher than the normal range compromising the crossover formation critically.Figure 76. The current study suggests that the meiotic checkpoint eliminates heat-damaged spermatocytes. This observation echoes the fact that most mammals cool their testes in the scrotum or through other mechanisms78. We propose that the meiotic checkpoint serves as a safety device, ensuring that only spermatozoa developed at low temperatures can fertilize eggs. Future investigations are warranted to understand the evolutionary constraint to cool the testes and eliminate damaged germ cells.The meiotic checkpoint is an evolutionarily conserved surveillance mechanism that eliminates damaged spermatocytes in various conditions. These include the meiosis block in interspecific hybrids, which leads to hybrid sterility, a vital means of speciation48. Alternatively, the amount of RPAs, RAD51, DMC1, or other proteins can be reduced at high temperatures. Furthermore, the foundation of the repair foci assembly provided by the axis, synaptonemal complex, and aligned chromosomes may be temperature-sensitive, as observed in other organisms, including Arabidopsis and C. elegans80. However, these factors also play roles in oogenesis, which occurs at the body core temperature in embryonic ovaries81 and at 37 °C in vitro as seen in a recently established culture using pluripotent cell-derived PGC-like cells83. Therefore, male-specific factor(s) must be crucial for temperature sensitivity, for example, sex-specific components of the DSB repair machinery or structures of the synaptonemal complex. Such sex dependence contradicts the possibility that the function of RPAs, RAD51, or DMC1 proteins per se is temperature-sensitive.The molecular mechanism underlying this delicate temperature sensitivity remains unclear. However, we would hypothesize some mechanisms explaining the meiotic failure at 38 °C and, to a weaker extent, at 37 °C. The findings of this study suggest that the increase in DSBs in leptotene and zygotene is the primary cause of meiotic failure at high temperatures. This appears to be, at least in part, due to the defective assembly of the DSB repair machinery on the DSBs, given the reduced RPA2, RAD51, and DMC1 foci. The potential underlying heat-sensitive processes include DNA resection generating single-stranded DNA, RPA binding to single-stranded DNA, and interaction between these proteinsC. elegans57. In mice, while LINE1 is unlikely to contribute to the temperature-dependent increase in DSBs, the involvement of other transposons is an open question that warrants further investigation. The assembly of MLH1+ late recombination nodules or crossover formation might also be heat-sensitive. More generally, temperature effects may alter over time between the initial (acute) and late (chronic) phases owing to the hormesis effect. Thorough and careful investigations are necessary to fully understand the mechanisms of heat-induced meiosis failure.Although defects in DSB repair are crucial for heat-induced meiotic failure, additional mechanisms may also be involved. In particular, the generation of DSBs can increase at high temperatures. In this regard, a considerable possibility is the dysregulation of transposons by heat, as recently demonstrated in By nurturing spermatogenesis at arbitrary temperatures, free from extratesticular factors, explant culture provides an invaluable and otherwise unavailable opportunity to study heat vulnerability in spermatogenesis. At the same time, the current culture supports spermatogenesis less efficiently than in vivo development, even at the optimum temperature (34 °C), which could modify the effect of temperature. Such inefficiency may reflect the subcritical defects observed at 34 °C, including expansion of LINE1 expression, a mild increase in DSBs, and a slight decrease in crossovers. These findings may help further optimize culture conditions and improve the usefulness of the explant culture.In conclusion, we hope that ex vivo culture and the findings of this study will help to better understand the mechanisms of temperature sensitivity in mammalian spermatogenesis. In addition to developing an infertility treatment strategy, the results would also provide insights into finding the answer to the fundamental question of why does mammalian spermatogenesis proceeds at low temperatures.Acr-GFP transgenic mice, designated formally as B6;B6C3-Tg(Acro3-EGFP)01Osb 22, with a genetic background of C57BL/6 J (Japan SLC and Japan CLEA), were used for histological and immunohistological analysis and ex vivo organ culture experiments. C57BL/6 J mice were used for temperature measurements and histological and immunohistological analysis of testes. All mouse experiments were conducted with approval from the Institutional Animal Care and Use Committee of the National Institutes of National Sciences.13.Eight-week-old C57BL/6 J mice were subjected to operations of artificial cryptorchidism following a rigorous method wherein the testes were wrapped by the associated fat pad and positioned deep in the abdomenIPPT-300 transponders (BioMedic Data Systems) were implanted into the scrotal sac and peritoneal cavity (under the liver) of wild-type C57BL6/J mice. One week after surgery, the temperature at each position was measured separately using a DAS-7006S scanner (BioMedic Data Systems) every 2 h for 24 h. Ambient conditions were maintained under a 12 h light/12 h dark at room temperature (25 ± 2 °C).85 with minor modifications. Briefly, 1.5% (wt/vol in PBS) agarose gels  were prepared and submerged in the culture medium αMEM/10% KnockOut Serum Replacement/1% Penicillin–Streptomycin) for >24 h, repeatedly 2–3 times. Whole testes dissected from euthanized neonatal Acr-GFP mice (4 days postpartum) were removed from the tunica albuginea and, without cutting into multiple pieces, gently positioned on the agarose gel in a 12-well culture plate with 500 µL of the above-described medium. The medium was changed once a week for 5 weeks. The culture incubators  were supplied with 5% CO2 in the air and maintained at specific temperatures (ranging from 30 to 40 °C), controlled with an accuracy of ±0.2 °C.Ex vivo culture was performed as previously describedThe grading of spermatogenesis progression based on Acr-GFP expression was performed visually at the time of weekly medium change, using an IX73 inverted fluorescence microscope (Olympus). Grading was performed unblinded as the observation had to be completed in a short period to minimize the temperature fluctuation.Testes removed from euthanized mice or cultured testis explants were fixed overnight in Bouin’s solution or 4% PFA in PBS at 4 °C, embedded in paraffin, sectioned, and mounted on a glass slide. According to standard protocols, sections were stained with hematoxylin and eosin (HE) or hematoxylin and periodic acid-Schiff (H-PAS) and mounted for microscopy.86. Briefly, the sections were incubated with Blocking One Histo  solution for 1 h at room temperature, followed by incubation with primary antibodies (described below) in 4% donkey serum/Can Get Signal Solution 1 (Toyobo) overnight at 4 °C. After the sections were washed with 0.04% Tween 20 in PBS, they were incubated with appropriate Alexa Flour-conjugated secondary antibodies (Thermo Fisher Scientific and Jackson ImmunoResearch) in 4% donkey serum/Can Get Signal Solution 2 containing Hoechst 33342 for 2 h at room temperature. After the slides were washed with the secondary antibody solution, they were mounted using Fluoro-KEEPER Antifade Reagent  for fluorescence microscopy.Immunofluorescence (IF) was performed on paraffin sections (7 μm thick) for most cases. For double staining of LINE1-ORF1 and SCP3  ; anti-KIT goat monoclonal antibody , anti-mouse Vasa homolog (MVH) rabbit polyclonal antibody ; anti-SCP3 mouse monoclonal antibody , anti-GATA4 rabbit polyclonal antibody ; anti-cleaved Caspase-3 rabbit polyclonal antibody ; and anti-LINE1-ORF1 rabbit polyclonal antibody . Cells were then suspended in PBS for hypotonic treatment by mixing with an equal volume of 30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, and 0.5 mM dithiothreitol. Cells were washed and resuspended in PBS, mixed twice with 100 mM sucrose solution (pH 8.2), and then with three volumes of 1% PFA/0.1% (v/v) Triton X-100 in 10 mM sodium borate, before being dropped onto a glass slide. The slides were then incubated in a humidified chamber for 1 h at room temperature, and then air-dried and frozen at −80 °C before IF. To obtain sufficient amounts of starting materials to avoid the massive loss because of their small size during these procedures, several  ex vivo explants were pooled.The nuclear spread of spermatocytes from in vivoFor IF, the specimens were first blocked with 10% or 2% fetal bovine serum in 0.05% Triton X-100 in PBS (blocking buffer) for 1 h at room temperature and then with primary antibodies (listed below) in blocking buffer overnight at 4 °C. The specimens were then washed with 0.05% Triton X-100 in PBS, and incubated with Alexa Flour-conjugated secondary antibodies diluted in blocking buffer containing Hoechst 33342 for 2 h at room temperature before they were washed and mounted for fluorescence microscopy.The primary antibodies used are as follows: anti-DMC1 rabbit polyclonal antibody , anti-SYCP1 polyclonal antibody , anti-SYCP3 rabbit polyclonal antibody , anti-SCP3 mouse monoclonal antibody , anti-phospho-H2A.X , anti-RPA2 , anti-RAD51, anti-MLH1 , and anti-HORMAD1   were observed and photographed using a BX51 upright fluorescence microscope equipped with a DP72 CCD camera operated using a cellSens imaging software (Olympus). Observations and signal quantification of γH2AX staining Fig.  were pert-test (p = 4.6 E-11).Figure Figure Figure Figure 89, with cells classified visually in the leptotene, zygotene, pachytene, or diplotene stage based on the SCP3 staining pattern. Four testes from different individuals were pooled for the in vivo sample, whereas cells prepared from 14 (34 °C), 15 (37 °C), and 15 (38 °C) explants were pooled for each ex vivo sample to prepare the nuclear spread specimens. As indicated in the panel, the number of spermatocytes analyzed to be in the leptotene stage was 73, 59, 79, and 98, those in the zygotene stage were 54, 62, 76, and 49, those in the pachytene stage were 89, 85, 71, and 0, and those in the diplotene stage were 68, 47, 45, and 0 for the in vivo and ex vivo explants at 34, 37, and 38 °C, respectively. In the dot plots, the values of the γH2AX signal are shown in the AU. Multiple comparisons were conducted using the Kruskal–Wallis test among all values within each category, providing p-values of 2.31E-34, 1.13E-19, 2.53E-19, and 3.76E-5 for leptotene, zygotene, pachytene, and diplotene, respectively, and using the Steel–Dwass test for combinations of two values within each category, resulting in p-values as indicated in the plots .Figure Acr-GFP mice and testis explants cultured for five weeks at 34, 37, or 38 °C, followed by immunostaining for RPA2 (5b), RAD51 (6b), or DMC1 (6d) and co-staining with SCP3. Photomicrographs were taken using a BX51 upright fluorescence microscope equipped with a DP72 CCD camera operated using cellSens imaging software (Olympus). Dots of RPA2 (5b), RAD51 (6b), or DMC1 (6d) associated with SCP3+ axes were counted manually, and each spermatocyte nucleus was classified to be in the early leptotene, late leptotene, zygotene, or pachytene stage. Cells prepared from three, four, and two testes from different individuals (in vivo) as well as 8, 14, and 4 (34 °C), 8, 15, and 10 (37 °C), and 8, 15, and 4 (38 °C) explants were pooled and analyzed in (5b), (6b), and (6d), respectively. The number of spermatocytes examined is indicated in the panels (N.D. not detected). Multiple comparisons were conducted using the Kruskal–Wallis test among all values within each category, with p-values of 0.88, 1.39E-3, 1.13E-11, and 0.02 in (5b), 0.01, 4.75E-18, 1.68E-9, and 0.11 in (6b), and 0.02, 9.08E-9, 2.20E-3, and 0.25 in (6d), respectively, for early leptotene, late leptotene, zygotene, and pachytene stage. The Steel–Dwass test was applied for combinations of two values within each category, resulting in p-values as indicated in the plots .Figures + chromosome axes and the absence of SCP3+/SCP1+ synapsed regions. Normal zygotene spermatocytes were those classified as zygotene spermatocytes  but not as aberrant pachytene described below. Aberrant pachytene spermatocytes were in an unusual state of zygotene , harboring one or more fully synapsed chromosomes and one or more autosome pairs that were unsynapsed or were partly synapsed in less than 50% of its length. Pachytene spermatocytes were defined authentically when all chromosomes had fully synapsed except for the XY body. The total number of spermatocytes counted was 495, 334, 330, and 176 for in vivo-developed testes  and testis explants cultured for five weeks at 34 °C (10 explants), 37 °C (15 explants), and 38 °C (10 explants), respectively. The resultant counts of leptotene, normal zygotene, aberrant pachytene, and pachytene spermatocytes were 49, 36, 3, and 407 in in vivo-developed testes and 45, 37, 27, and 225 in explants at 34 °C, 113, 44, 134, and 39 in explants at 37 °C, and 113, 36, 27, and 0 in explants at 38 °C, respectively.Figure + axes were counted manually in spermatocytes harboring at least one fully synapsed chromosome [including aberrant pachytene and (fully synapsed) pachytene spermatocytes], on chromosome spread stained for MLH1 and SCP3. Cells prepared from three testes of different individuals and those from 10 (34 °C), 12 (37 °C), and 12 (38 °C) explants were pooled and analyzed. The total number of spermatocytes counted were 158, 108, 158, and 50, respectively.Figure p-values of 9.53E-13, and Steel–Dwass test for combinations of two values, resulting in the p-values as indicated in the panel.Figure p < 0.01 between scrotal and abdominal temperatures based on statistical evaluation using a matched Student’s t-test.Supplementary Fig. + spermatocytes of those with SCP3+ spermatocytes was counted in the in vivo-developed 5-week-old mouse testes, artificial cryptorchid testes (2 days after surgery), and testis explants cultured for five weeks at 34, 36, 37, and 38 °C. Averaged percentage ± SEM calculated from five in vivo testes, five artificial cryptorchid testes, and 5 (34 °C), 5 (36 °C), 6 (37 °C), and 4 (38 °C) explants, respectively. Student’s t-test was used to determine p-values as indicated .Supplementary Fig. All calculations and statistical analyzes were performed using Microsoft Excel 2019, KaleidaGraph, Bell Curve for Excel, and R (Supplementary Table Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Ziziphus jujuba Mill.) has attracted increasing attention because of its fruits’ high nutritional and medicinal value, which produce pentacyclic triterpenoids with valuable pharmacological activities beneficial to human health. However, the dynamic accumulation and metabolism pathway of triterpenoids remain unknown in jujube. Here, we performed metabolite assays of triterpenoids and expression analysis of genes involved in the corresponding metabolic processes on cultivated jujube (Z. jujuba cv. Junzao) and one type of wild jujube (Z. jujuba var. spinosa cv. Qingjiansuanzao). Our results showed that the triterpenoids accumulate predominantly in young leaves, annual stems, buds, and white-mature and beginning red stage fruit. Besides, the total triterpenoid content, ceanothic acid, oleanonic acid, and 3-ketoursolic acid were higher in ‘Qingjiansuanzao’ than in ‘Junzao’. Moreover, we found 23 genes involved in terpenoids metabolism were expressed in all organs, and the ZjSQS1, ZjCYP450/1, ZjCYP450/3, ZjOSC1, ZjFPS, and ZjAACT2 gene expression patterns were consistent with metabolites accumulation during fruit development. In addition, 100 μM MeJA induced ZjSQS1, ZjFPS, and ZjHMGR3 expression in leaves and enhanced triterpenoids accumulation. These findings will help understand the unique metabolism of terpenoids and will benefit further utilization and breeding of jujube as both edible fruit and functional food.Jujube ( Ziziphus jujuba Mill.) and wild jujube (Z. jujuba Mill. var. spinosa Hu.) are two representative species and subspecies of the Rhamnaceae family [Jujube , which is involved in plant defense, usually performs as a signaling molecule to resist different biotic and abiotic stresses , and plae family ,19,20,21Hence, triterpenoids continue to be of interest because of their pharmacological properties and biological activities. This study aimed to explore triterpenoid metabolites’ spatial and temporal distribution in a jujube cultivar ‘Junzao’ and a wild jujube accession ‘Qingjiansuanzao’. We conducted genome-wide identification of triterpenoid metabolic pathway genes. Transcriptional expression analysis revealed triterpenoid metabolic-associated gene expression in different tissues and developmental stages. We also analyzed the induction of MeJA on triterpenoid synthesis. This study highlights spatial metabolic accumulation characteristics and candidate genes underlying jujube’s triterpenoid metabolism. The findings lay a foundation for revealing the unique terpenoid metabolic pathway of jujube. It will help understand the unique metabolism of terpenoids and provide a reliable basis for resource utilization and breeding of jujube as both edible fruit and functional food.The jujube cultivar ‘Junzao’ and a wild jujube ‘Qingjiansuanzao’ were grown at the Jujube Experimental Station of Northwest A&F University in Qingjian, Shaanxi, China. Fruits of different developmental stages at 30, 50, 80, 90, 100, and 110 d after pollination (DAP) were carefully collected. These stages were designated as young fruit (YF), enlargement (EF), white mature (WM), beginning red (BR), half red (HR), and full red ripening stages (FR), respectively. Samples of different tissues, flowers (Fl), buds (Bd), young leaves (YL), mature leaves (ML), and stems (St) were collected. The plant tissues and fruit samples (at least 20 fruits per sample) were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent analysis. ‘Qingjiansuanzao’ jujube seedlings were grown in a light incubator under 16 h light (24 °C) and 8 h dark (18 °C) cycles.v/v) methanol/H2O. The mixture was ultrasonicated for 40 min and stored at 4 °C overnight. The supernatant was collected for further analysis. Total triterpenes content (TTC) was determined according to Wei et al. . FirstlyA total of 20 µL plant extract or standard ursolic acid  (5–40 µg) and 150 µL acidic vanillin reagent  was mixed. Perchloric acid (500 µL) was added to the reaction mixture and heated in a water bath for 15 min at 60 °C. Further, all reaction mixtures were placed on an ice bath at room temperature, and equal to 2.25 mL of glacial acetic acid was added. Absorbance was measured at 548 nm and results were calculated as mg ursolic acid equivalents (UAE)/g extract.Freeze-dried jujube fruit samples (approximately 0.5 g) were extracted with 5 mL of 90% MeOH in an ultrasonic bath for 35 min, mixed well, placed at 4 °C overnight in the dark, and centrifuged at 12,000 rpm for 10 min. The supernatants were filtered through a 0.22 μm nylon filter film for high-performance liquid chromatography (HPLC) analysis following Guo et al. with some modifications . The extThe HPLC system  was equipped with a C18 analytical column . The mobile phases were (A) 100% MeOH and (B) 0.5% ammonium acetate in water. The elution gradient established was 83% A and 17% B for 45 min. The temperature was controlled at 30 °C, and the flow rate was 0.8 mL/min with an injection volume of 10 μL. The post-run time was 10 min, and the monitoring of triterpenoids acid was carried out at 210 nm. The mixed standard solution  was prepared using MeOH.The triterpenoid acid compounds were identified by comparing the HPLC retention times with those of the standards. The triterpenoids quantification was based on a linear calibration diagram of the peak areas’ logarithm versus the concentration’s logarithm.® Premix Ex TaqTM II . The reaction procedure was as follows: 1 cycle at 98 °C for 30 s, 40 cycles at 95 °C for 5 s, 56 °C for 30 s, and finally 72 °C for 30 s. The expression levels were analyzed using the 2−ΔΔCT method using the ZjUBQ1 and ZjUBQ2 genes as internal standards [Total RNA was isolated using a Plant RNA extraction kit . The concentration was measured using a NanoDrop-2000 . The first-strand cDNA was synthesized using 200 ng total RNA following the Prime Script TM RT reagent kit protocol with a gDNA Eraser . The qRT-PCR analysis was performed using a LightCycler 96 assay system . According to the manufacturer’s protocol, all reactions were performed using the TB Greentandards ,25. All Leaves of ‘Qingjiansuanzao’ jujube seedlings were harvested after 100 μM MeJA-treatment or water control. MeJA solution and the distilled water were sprayed on leaf surfaces until saturation. Samples were collected at 0, 12, 24, 36, 48, 60, 72, 84, and 96 h after treatment, frozen in liquid nitrogen, and stored at −80 °C for further experiments.p < 0.05). Two-tailed Pearson’s correlation coefficients were analyzed to delineate the correlation between synthetic genes expression level and triterpenoid accumulation. The pictures were illustrated using TBtools software and OmicShare Tools online: https://www.omicshare.com/tools/ (accessed on 13 February 2022).The significant differences were calculated by the analysis of variance (ANOVA) and Duncan’s multiple range test  C. MoreovTo better understand the metabolic accumulation patterns of different triterpenoids in tissues and the developmental stages of ‘Qingjiansuanzao’ and ‘Junzao’, four types of the seven major pentacyclic triterpenoids  were determined by HPLC . PCA anaIn different tissues, except ceanothic acid, six triterpenoids mainly accumulated Bd and YL of ‘Qingjiansuanzao’ and ‘Junzao’. Moreover, ceanothic acid was mainly concentrated in St with relatively higher than other tissues . The resFor the different fruit development stages, the variation in triterpenoid content increased significantly before the WM development stage, and then decreased for all triterpenoids except tannic acid. Among different stages, the WM and BR were the key synthesis stages of pentacyclic triterpenoids. In addition, the triterpenoid contents were higher in FR than in the YF development stage B. We theThrough previous transcriptomic data analysis , we idenZjAACT1, ZjAACT2, and ZjAACT3 mRNA levels increased with fruit development after the YF stage, and there were similar trends in ‘Qingjiansuanzao’ and ‘Junzao’. ZjHMGS1, ZjHMGS2, ZjSQE1, ZJSQE2, ZjSQE3, ZjOSC2, and ZjUGT1 had higher expression in YF and EF of jujube stage, suggesting these genes play a key role in triterpenoids at the early stage. ZjHMGR1, ZjHMGR3, ZjSQS1, ZjSQS2, ZjOSC1, ZjP450/1, ZjP450/2, ZjP450/3, ZjUGT2, ZjUGT3, and ZjUGT4 expressions were higher in WM and BR stages, ZjFPS and ZjHMGR2 expression patterns were consistent with the development of jujube, showing a decrease and then an increase along with fruit development.ZjSQS1, ZjP450/3, and ZjP450/1 . Besides, ZjAACT1, ZjFPS, ZjOSC1, ZjAACT2, and ZjSQS2 are also highly correlated with corosolic acid, betulinic acid, and ursolic acid. Simultaneously, the expression of ZjP450/2, ZjOSC2, ZjAACT3, ZjHMGR1, and ZjHMGR3 were positively correlated with the corosolic acid, oleanolic acid, oleanonic acid, and 3-ketoursolic acid contents. However, the ceanothic acid, oleanonic acid, and 3−ketoursolic acid were weakly correlated with the expression of ZjSQS1, ZjP450/1, and ZjP450/3 and negatively correlated with ZjAACT3 and ZjHMGR1. Moreover, ZjSQE1, ZjSQE2, ZjSQE3, ZjHMGS1, ZjHMGS2, and ZjHMGR2 expressions were negatively correlated with seven triterpenoids. In contrast, the ZjSQS1, ZjP450/1, and ZjP450/3 expressions were positively correlated with most triterpenoids content. In general, the ZjSQS1, ZjP450/1, ZjP450/3, ZjOSC1, ZjFPS, and ZjAACT2 were highly correlated with triterpenoid content, which suggested that these genes might be key genes in triterpenoid synthesis.To explore the relationship between triterpenoids and candidate genes, we correlated the content of seven triterpenoids and the expression of genes controlling triterpenoids synthesis A. CorrelWe selected genes showing high correlations (Pearson correlations > 1.00) between metabolite accumulation and gene expression A. To conZjFPS, ZjSQS1, ZjSQS2, and ZjP450/3 had slightly higher expression in ‘Qingjiansuanzao’ than in ‘Junzao’  of ‘Qingjiansuanzao’ and ‘Junzao’. In addition, the overall expression level of ZjSQS1 and ZjP450/3 was high during fruit development. Analysis of the candidate genes expression levels in ‘Qingjiansuanzao’ and cultivated jujube showed that ZjAACT2, ZjHMGR3, ZjSQS1, ZjOSC1, and ZjOSC2 were highly expressed in ‘Qingjiansuanzao’ jujube. In contrast, ZjFPS, ZjSQS2, and ZjP450/3 genes were highly expressed in cultivated jujube, suggesting that these genes may be responsible for triterpenoid metabolism differences.Gene expression of The triterpenoid biosynthesis was further analyzed to explore further the response of triterpenoid synthesis genes and triterpenoid metabolites to MeJA induction . In respZjHMGR3, ZjFPS, and ZjSQS1 were upregulated after MeJA treatment for 84 h  is widely applied to medicinal plants to promote triterpenoids and other secondary metabolites. Exogenously applied elicitor MeJA stimulates the biosynthesis of many secondary metabolites , and MeJ lucidum ,38. MeJAnd birch . It is wnd birch ,41,42. WZjAACT1, ZjFPS, ZjSQS1, ZjOSC1, and ZjP450/3 were identified as key candidate genes for triterpenoid synthesis. Moreover, triterpenoid acid metabolites and genes respond to the MeJA induction. These studies deepened the systematic and comprehensive understanding of triterpenoid metabolism in jujube, laying a foundation for breeding for fruit quality and bioactive functions.Our study elucidated the spatial metabolism pattern of pentacyclic triterpenes in the wild jujube ‘Qingjiansuanzao’ and the cultivated jujube ‘Junzao’, ceanothic acid, oleanonic acid, and 3-ketoursolic acid were highly accumulated in wild jujube, while the betulinic acid, corosolic acid, and ursolic acid were major pentacyclic triterpenoids found in cultivated jujube. In addition, the triterpenoids accumulated mainly in young leaves, annual stems and buds, and in the middle and late stages (after the EF stage) of fruit development. From 23 identified genes from transcriptome data,


These results suggest that fentanyl exposure during embryonic development is neurotoxic, highlighting the importance of zebrafish as an aquatic species in research on the neurobehavioral effects of opioids in vertebrates.The use of fentanyl during pregnancy, whether by prescription or illicit use, may result in high blood levels that pose an early risk to fetal development. However, little is known regarding the neurotoxicity that might arise from excessive fentanyl exposure in growing organisms, particularly drug-related withdrawal symptoms. In this study, zebrafish embryos were exposed to fentanyl solutions  for 5 days post fertilization (dpf), followed by a 5-day recovery period, and then the larvae were evaluated for photomotor response, anxiety behavior, shoaling behavior, aggression, social preference, and sensitization behavior. Fentanyl solutions at 1 and 5 mg/L induced elevated anxiety, decreased social preference and aggressiveness, and behavioral sensitization in zebrafish larvae. The expression of genes revealed that embryonic exposure to fentanyl caused substantial alterations in neural activity ( Opioids, a group of alkaloids and derivatives isolated from the opium poppy plant, are frequently used to treat cancer pain, post-operative pain from surgery, and other types of chronic pain. In the past two decades, drug dependence and addiction due to the nonmedical use of opioids have been identified as a significant public health problem .Chronic accumulation of toxic opioid metabolites in adults can result in opioid-induced neurotoxicity, which includes hallucinations, delirium, confusion, seizures, and nociceptive hypersensitivity . AdditioThe zebrafish (Danio rerio) has been widely employed in genetic and developmental research in recent years . With thμ-receptor affinity and gamma aminobutyric acid (GABA) agonist actions, which results in a complex neurotoxic mechanism . The Trizol reagent was acquired from Jiancheng . Reverse transcriptase kits and SYBR green reagents were purchased from Takara .Fentanyl hydrochloride powder  was purchased from Shanghai Yuansi Standard Science and Technology Co., Ltd. All other reagents and chemicals were purchased from Sigma-Aldrich . Fentanyl hydrochloride powder was dissolved in distilled water and then diluted to 0.1, 1, 5 mg/L in E3 medium , and the room was lit for 14 h and dark for 10 h (lights on at 8:00 am). Fertilized eggs were collected by generating spawning activity in adult zebrafish. The night before the experiment, male and female zebrafish were positioned in a tank in a 2:1 ratio, separated by a transparent acrylic panel. The next morning at 8:00 am, the divider was removed to trigger natural spawning under the stimulation of light, and eggs were collected within 1 h. Normally developed embryos were chosen for tests following standard techniques . The lar2, and 0.33 mM MgSO4) and placed in standard 90 mm Petri dishes within 2  h of fertilization . Petri dishes were kept in the incubator at a 14-hour light: 10-hour dark cycle, with 15 mL of exposure solution replaced daily . After the 2 hpf to 5 days post-fertilization (dpf) exposure test, all zebrafish larvae in the fentanyl and control groups were transferred to E3 solution to begin a five-day recovery period and fed once daily until 14 dpf. Larvae that completed the behavioral tests . The bottom of the 24-well plate was covered with a black and white acrylic plate, which divided the circular wells into two large equal areas of light and dark zones . The DanFollowing methods previously reported , we examAt 12 dpf, 12 larvae were taken at random from each fentanyl concentration group and transferred to rectangular 6-chamber polypropylene dishes  for mirror-attack studies. A mirror paper (3.5 × 1.5 cm) was placed to one side of each chamber (3.5 × 3.5 × 1.5 cm) of the dish . Zebrafi s and quickly transferred to 96-well plates (300 µL of 0.01 mg/L fentanyl solution per well). The behavior of the larvae was monitored. Once the zebrafish larvae had acclimated to the dark for 10 min, their responses to three successive 10-minute light (a light intensity of 100 lux) and 10-minute dark periods were studied. The swimming distances of larvae during light (visible light) and dark (infrared light) periods were examined to determine the behavioral effects of repeated exposure of larvae to the fentanyl.At 14 dpf, 12 zebrafish larvae from each concentration group were randomly moved to a 0.01 mg/L fentanyl solution with 10 min bdnf, c-fos) as well as neuronal development and plasticity  were examined in zebrafish larvae at 5 dpf after fentanyl exposure. Thirty larvae were randomly selected from each concentration group and total RNA was extracted using Trizol reagent  after hypothermia anesthesia.RNA was quantified by measuring the absorbance at 260 nm and diluted to 200 ng/L. Takara’s cDNA first strand synthesis kit  was then used to reverse-transcribe 1 µg of total RNA. Following the manufacturer’s protocol, quantitative analysis by qPCR was performed using the CFX-96™ Real-Time PCR Detection System . The cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. β-actin expression levels were used to normalize the relative expression ratios of neural-related genes, which were then evaluated using the 2−(ΔΔCt) method  was used to investigate differences between the treatment and control groups. The normality of the behavioral data was tested using Kolmogorov-Smirnov, and the homogeneity of variance was tested using Bartlett’s test. Welch’s ANOVA was used when the data did not meet the assumption of homogeneity of variance. Each treatment was compared to the control group using the appropriate post hoc test (Dunnett’s T3 multiple comparison test) when differences were found to be statistically significant. Prism 9  was utilized to analyze significant differences, and statistical significance thresholds for all experiments were set at *p < 0.0001; Dunnett’s T3 post hoc test, all p < 0.0001) . Only the group that was given 0.1 mg/L of fentanyl did not experience a significant reduction in travel distance during the dark phase in comparison to the control larvae .The behavioral capacity of zebrafish larvae at 5 dpf was evaluated by the distance traveled under light and dark stimuli. The findings indicated that zebrafish larvae moved farther during the dark period than they did during the light period . Fentanyl larvae  (one-wayp = 0.1808), zebrafish that had been exposed to 1 or 5 mg/L fentanyl up to 5 dpf and then recovered for 5 days, traveled significantly smaller distances than control fish in the dark conditions .Fentanyl continued to impair the behavioral abilities of zebrafish larvae after a 5-day recovery interval . Althougnditions   and made significantly fewer entrances into the central region . Another important indicator of high levels of anxiety in zebrafish larvae is the increased preference for light . Also, compared to the control group, the larvae in the 1 mg/L and 5 mg/L fentanyl groups moved between the light and dark areas significantly more often .When exposed to fentanyl for 5 dpf and then recovered for 5 days in the clean medium, zebrafish larvae showed a decrease in their preference for the central region (thigmotaxis), which indicated increased anxiety. Compared to control fish, 10 dpf larvae exposed to fentanyl spent significantly smaller time in the central region   and increased nearest neighbor distance (NND)  = 5.363, p = 0.0023; Dunnett’s T3 post hoc test, p = 0.0038 for 1 mg/L, p = 0.0023 for 5 mg/L) compared to control fish . Although the fentanyl-exposed fish spent more time in the mirror region than control fish, the differences were not significant (p = 0.4818).For shoaling at 11 dpf, zebrafish exposed to 1 or 5 mg/L fentanyl showed significantly increased inter-individual distance (IID)  = 8.934, rol fish . Zebrafirol fish   and dark periods .Behavioral sensitization refers to the enhanced motor stimulus response that occurs during repetitive, intermittent exposure to a specific drug, and in the present study we evaluated this by the motor ability of zebrafish in photomotor response. The locomotor response to a repeated fentanyl stimulus at 14 dpf was studied in zebrafish that had been exposed to fentanyl at 0.1, 1 or 5 mg/L during the first 5 dpf, then recovered in clean E3 medium up to 14 dpf. Zebrafish larvae exposed to fentanyl during development experienced behavioral changes after re-exposure to fentanyl . After t periods   and neuronal development and plasticity . At 5 dpf, fentanyl exposure at a concentration of 5 mg/L significantly increased the relative expression of bdnf  = 9.878, p = 0.0046; Dunnett’s T3 post hoc test, p = 0.0181), c-fos  = 5.327, p = 0.0261; Dunnett’s T3 post hoc test, p = 0.0172), napas4a  = 23.49, p = 0.0003; Dunnett’s T3 post hoc test, p = 0.0003), egr1  = 12.199, p = 0.0024; Dunnett’s T3 post hoc test, p = 0.0090) in comparison to control larvae  = 76.72, p < 0.0001; Dunnett’s T3 post hoc test, p = 0.000 for 1 mg/L, p < 0.0001 for 5 mg/L) and ier2a  = 11.51, p = 0.0028; Dunnett’s T3 post hoc test, p = 0.0116 for 1 mg/L, p = 0.0013 for 5 mg/L) genes relative to controls .We further examined how different concentrations of fentanyl affected the transcriptional regulation of genes regulating neural activity  test, the lethal concentration of fentanyl was 83.17 µM (28.40 mg/L) and the sublethal value  was 16.15 µM (5.52 mg/L) in 5 dpf zebrafish larvae . The rani.e. light inhibits behavior and dark promotes behavior, which needs supraspinal input from the brain and is a crucial sign for determining neurodevelopment (Oprm1 (Opioid receptor mu1) expression is detectable in numerous nerve centers at 24 h post fertilization in zebrafish embryos  play critical roles in neuronal cell proliferation, differentiation, and maintenance, as well as synaptic plasticity, learning, and long-term memory. The neurotrophic factor bdnf has been identified as a possible downstream target of c-fos and npas4a  and ier2a are key regulators of vertebrate neural development , a neuropeptide involved in crucial neural processes such as synaptic plasticity, neurogenesis, and brain neurite growth, is produced by brain-derived neurotrophic and nerve growth factors  and neuronal development and plasticity . The results demonstrated the neurotoxicity of fentanyl at nonteratogenic concentrations to developing organisms, providing evidence for the risk of fentanyl use during pregnancy.In conclusion, we reveal for the first time that exposure of zebrafish embryos to fentanyl resulted in elevated anxiety, decreased social preference and aggressiveness, and induced behavioral sensitization. The expression of genes showed that embryonic exposure to fentanyl at concentrations of 5 mg/L caused substantial alterations in neural activity (10.7717/peerj.14524/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14524/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14524/supp-3Supplemental Information 3Click here for additional data file.


Pyrus communis L.) and it’s mutants were used to explore the mechanism that affects branch formation by conducting phenotypic trait assessment, hormone content analysis, and transcriptome analysis. The mutant plant (MP) showed fewer branches, shorter 1-year-old shoots, and longer petiole length, compared to original plants (OP), i.e., wild type. Endogenous hormone analysis revealed that auxin, cytokinin, and jasmonic acid contents in the stem tips of MP were significantly higher than those of the original plants. In particular, the jasmonic acid content of the MP was 1.8 times higher than that of the original plants. Transcriptome analysis revealed that PcCOI1, which is a transcriptional regulatory gene downstream of the jasmonic acid signaling pathway, was expressed more highly in the MP than in the original plants, whereas the expression levels of PcJAZ and PcMYC were reduced in the MP compared with that of the original plants. In response to treatment with exogenous methyl jasmonate, the original plants phenotype was consistent with that of the MP in developing less branches. These results indicate that jasmonic acid negatively regulates branch growth of pear trees and that jasmonic acid downstream regulatory genes play a crucial role in regulating branching.The quality of seedlings is an important factor for development of the pear industry. A strong seedling with few branches and suitable internodes is ideal material as a rootstock for grafting and breeding. Several branching mutants of pear rootstocks were identified previously. In the present study, ‘QAU-D03’ ( Pear rootstock with a strong stem, few branches, and suitable internodes is ideal material for grafting and breeding. Branch development and growth are regulated by a complex network of factors involving plant growth and development, hormone regulation, transcriptional regulation, and other factors.Bud mutation is an important means of causing changes in branching through plant somatic variation . The merSMALL AUXIN UPREGULATED RNA 10 (SAUR10), thus participating in the regulation of branching angle  proteins play an inhibitory role in the JA signaling pathway  gene encodes a SBP transcription factor, which is mainly expressed in meristems and is critical to regulating the branching and vegetative growth of alfalfa plants , is a member of the TCP family that negatively regulates rice tillering and inhibits the subsequent growth of axillary buds  affects cell proliferation, axillary bud growth, and branching of Arabidopsis thaliana. This gene encodes an AP2/ERF transcription factor and is highly expressed in proliferating cells  and less-branching mutants as materials. The results provide novel insights to improve understanding of the molecular mechanism of branch development in pear, which is of considerable importance for rootstock breeding.The growth characteristics of pear rootstock have important influence on grafting effect. Sturdy seedlings and few branches make ideal stock material. In this study, the mechanism affecting pear branching formation were explored through phenotypic trait assessment, hormone content analysis and transcriptomic analysis, by using pear ‘QAU-D03’ (22.1Pyrus communis L.). The shoots of OP were exposed to gamma radiation at 50 Gy  and then grafted onto one-year-old rootstocks of Pyrus betulifolia Bunge. Among the resulting trees, one less-branching mutant was found and named MP. One-year-old shoots of OP and MP plants were collected from the Jiaozhou Demonstration Park in Qingdao, Shandong Province, China, for tissue culture to obtain tissue-cultured plantlets. The plants were grown on MS medium with 1.0 mg·L-1 6-BA, 0.1 mg·L-1 IBA, 30 g·L-1 sucrose, and 7 g·L-1 agar in a tissue culture room. Tissue-cultured shoots with 20 days of uniform growth were selected for rooting. Once the shoots had produced 3-4 roots of length 2-3 cm, the plantlets were transplanted to a mixture of perlite:vermiculite:peat . Samples were collected at the shoot dormancy in mid-March and bud expansion stages at the end of April, with three biological replicates per sample, for transcriptome sequencing analysis.The original plant (OP) with more branches was a seedling progeny of ‘QAU-D03’  for 60 min at each step. Samples were placed in 100% ethanol and left overnight. After decoloration with dimethylbenzene, the samples were embedded in paraffin. Sections (8 μm) were cut using a rotary microtome , then dewaxed, rehydrated, cleaned, stained with toluidine blue, and the coverslip mounted with neutral balata. Sections were observed and photographed using an optical microscope . In addition, buds were observed by scanning electron microscopy. After manual removal of the bud scales, the buds were fixed, washed, and dehydrated as described. The material was dried by carbon dioxide critical point drying and sputter-coated with gold. The material was observed and photographed with a scanning electron microscope .2.4−1 Na2CO3, 2.93g·L−1 NaHCO3, and 0.02g·L−1 NaN3, pH 9.6) containing 0.25 μg·mL−1 antigens against the hormones. The coated plates were incubated for 4 h at 37°C and then kept at room temperature for 30–40 min. The plate was incubated for 3 h at 28°C for measurement of dihydrozeatin riboside (DHZR), zeatin riboside (ZR), brassinolide (BR), methyl jasmonate (JA-Me), indole-3-acetic acid (IAA), indolepyruvic acid (IPA), gibberellins (GAs), and overnight at 4°C for IAA, and then washed as described above. Color development in each well was detected using an ELISA Reader  at an optical density of A490. The contents of DHZR, ZR, BR, JA-Me, IAA, IPA, GAs, and ABA were calculated following the method of Fresh plant stem tips material (0.2-1.0 g) was ground in an ice-cooled mortar in 10 mL of 80% (v/v) methanol extraction medium containing 1 mM butylated hydroxytoluene as an antioxidant. The extract was incubated at 4°C for 4 h and then centrifuged at 4000 rpm for 15 min at 4°C. The supernatant was filtered through Chromosep C18 columns , and prewashed with 10 mL of 100% (w/v) and 5 mL of 80% (v/v) methanol. The hormone fractions eluted with 10 mL of 100% (v/v) methanol and 10 mL ether from the columns were dried under N2 gas, dissolved in 2 mL phosphate buffer saline containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5) for analysis by an enzyme linked immunosorbent assay (ELISA) . The ELI2.5Pyrus communis Bartlett DH Genome v2.0) and gene model annotation files were downloaded from GDR database (https://www.rosaceae.org/species/pyrus/pyrus_communis/genome_v2.0). An index of the reference genome was generated using HISAT2 v2.0.5 and paired-end clean reads were aligned to the reference genome using HISAT2 v2.0.5. FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. The fragments per kilobase of exon per million mapped fragments (FPKM) value of each gene was calculated based on the length of the gene and number of reads mapped to the gene. Differential expression analysis of two conditions/groups  was performed using the DESeq2 R package (v1.20.0). Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was implemented with the clusterProfiler R package for which gene length bias was corrected. Statistical enrichment of DEGs in KEGG pathways was detected with the clusterProfiler R package.Plant total RNA isolation kit  was used to extract total RNA from samples, following the manufacturer’s instructions, and different samples were subjected to three biological replicates. After qualification using a bioanalyzer , 1 μg of each sample was used for cDNA library construction. Four RNA sequencing libraries were constructed, in which MP1 and OP1 were the dormant shoot tips, MP2 and OP2 were the buds in expansion period. For library construction, 1 μg of RNA per sample was used as the input material. Library quality was assessed with a 2100 Bioanalyzer system . The clean dataset was obtained by removing reads containing the adapter sequence, poly-N, and low-quality reads from the raw data. The Q20, Q30, and GC content of the clean data were calculated. All downstream analyses used the high-quality clean data. The reference genome  and the ChamQ SYBR Color qPCR Master Kit (Vazyme Biotech Co.) were used to estimate relative gene expression levels under the different treatments. The reaction system  consisted of 2 μL template cDNA, 1 μL each forward and reverse primer, 10 μL Supermix, and 6 μL RNA-free water. The reaction protocol was as follows: 95°C for 5 min, then 45 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The Actin gene was used as an internal control. The relative expression level for each gene was calculated with the 2−ΔΔCt method. Each sample analysis was repeated three times. The primers used are listed in Total RNA was extracted from pear using the RNAprep Pure Plant Kit . The cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit . The Lightcycler2.7-1 Methyl jasmonate . After JA-Me treatment, observations were carried out after 10 days.The seedlings of ‘OP’ and ‘MP’ clones were selected and subcultured with Murashige and Skoog (MS) medium . The seedlings were divided into different treatments: (i) ‘OP’ and ‘MP’ control: continued use of the MS nutrient solution; (ii) ‘OP’ and ‘MP’ treated with JA-Me: MS with 100 μmol L2.8Statistical analysis was performed using IBM SPSS Statistics 23.0 . Values are presented as the mean ± SD of at least three independent biological replicates. The significance of differences between means was analyzed with Duncan’s multiple range test or Student’s t‐test. The probability level p < 0.05 was considered to be significant.33.1The wild type (OP) plants had more branches, whereas the mutant (MP) plants developed fewer branches and the leaves were larger Table 1.3.2The MP vascular bundle was larger than that of OP in the leaf, stem, and axillary buds at different development stages Table 2.3.3Endogenous hormone analysis revealed that the content of GA4 in OP was significantly higher than that in MP, whereas the contents of auxin (IAA), methyl jasmonate (JA-Me), indolepro pionic acid (IPA), trans-zeatin-riboside (ZR), brassinolide (BR), and dihydrozeatin-riboside (DHZR) in MP were higher than those in OP. The difference in JA-Me content between OP and MP was most marked Figure 33.4AP2/ERF, P450/CYP, MYB, WRKY, TCP, and NAC families  and TI2 are essential to preclude the proliferation of meristems and simultaneously produce thorns  BioProject database repository, accession number PRJNA916958.YC and CL conducted the experiment, and YC prepared the manuscript. ZQ, SZ, and JL provided important technical help in transcriptional analysis, YY, RW, and CM gave useful guidance and valuable discussion. JY, ZC, and JS provided the plant materials and important technical help in histological Observation. DL conceived the idea and provided financial support. All authors contributed to the article and approved the submitted version.


CHOP) gene expression. Simultaneously, 1 and 10 μg/mL TA significantly increased the proportion of normal fertilized oocytes  and blastocyst formation rate compared to the control, as well as that of 100 μg/mL TA after IVF by upregulating polyspermy-related genes. In conclusion, TA during IVM enhances 2PN and blastocyst formation rates by regulating organelles’ functions and activities.Tannin (TA) improves porcine oocyte cytoplasmic maturation and subsequent embryonic development after in vitro fertilization (IVF). However, the mechanism through which TA blocks polyspermy after IVF remains unclear. Hence, the biological function of organelles  and the incidence of polyspermic penetration were examined. We found no significant difference in oocyte nuclear maturation among the 1 µg/mL, 10 µg/mL TA, and control groups. Moreover, 100 μg/mL TA significantly reduced 1st polar body formation rate compared to the other groups. Additionally, 1 and 10 μg/mL TA significantly increased the protein levels of GDF9, BMP15, and CDK1 compared to the control and 100 μg/mL TA groups. Interestingly, 1 and 10 μg/mL TA improved the normal distribution of CGs, Golgi, ER, and mitochondria by upregulating organelle-related gene expression and downregulating ER stress ( The maturation process of mammalian oocytes is an extremely complicated biological event that includes both nuclear and cytoplasmic changes ,2. NucleTannins (TAs), powerful antioxidants, are a class of polyphenolic biomolecules found in a wide range of plants . MoreoveAll chemicals and reagents were obtained from Sigma-Aldrich Chemical Company  unless otherwise stated.2, 100% relative humidity (RH), and 38.5 °C for 42 h [Porcine ovaries were transported from the local slaughterhouse  to the laboratory at 30–34 °C in thermos, and cumulus–oocyte complexes (COCs) were obtained from small antral follicles (3–6 mm in diameter) using a syringe. Oocytes surrounded by a compact cumulus cell mass and homogeneous ooplasm were collected and washed with tissue culture medium-199  containing 10 mM HEPES, 0.3% polyvinyl alcohol (PVA), and 1% penicillin–streptomycin, and placed into culture dishes containing IVM medium  with 0, 1, 10, and 100 μg/mL TA . The pooled 50 COCs/condition was then cultured in an incubator at 5% COfor 42 h . After 46 spermatozoa/mL and placed into 50 μL mTBM droplet with matured oocytes. Immediately before insemination, sperm motility was assessed and >80% motile sperm was used in each experiment. Then, the samples were co-incubated for 6 h under 5% CO2 atmosphere at 38.5 °C. Subsequently, the presumptive zygotes were washed three times and transferred into an in vitro culture medium, porcine zygote medium 3 (PZM3), then incubated at 38.5 °C at 100% RH and 5% CO2 for 7 days. The zygotic pronucleus was detected by nuclear staining without the zona pellucida in 10 μg/mL Hoechst-33342 for 4 min after an additional 12 h of IVF. Moreover, cleavage and blastocyst rates were evaluated on day 2 and day 7, respectively. Blastocysts were collected on day 7 and stained with 10 μg/mL Hoechst-33342 for 4 min in order to count the total cell number per blastocyst.IVF was performed as described by Jin et al., with modifications . After 4Immunofluorescence staining was performed as previously described by Lee et al. . PorcineAfter 42 h of IVM, mature porcine oocytes were fixed with 4% PFA for 30 min, washed, blocked with 0.3% BSA and 1 M glycine solution, and permeabilized in 0.1% Triton X-100 for 5 min. After washing three times, the oocytes were incubated with 100 µg/mL fluorescein isothiocyanate conjugated to peanut agglutinin  for 30 min. The labeled oocytes were mounted, and the cortical granule (CG) distribution was observed under a laser-scanning confocal microscope . The distribution of CGs was classified as below , according to a method described previously . The actFor Golgi apparatus and endoplasmic reticulum (ER) staining, matured oocytes were fixed in 4% PFA for 10 min, washed three times with pre-cooled 0.2% PVA-PBS, and then placed in 5 mM NBD C6-Ceramide  and 1 μM ER-Tracker Red  for 30 min at 4 °C in the dark. The samples were blocked in 2% BSA-PBS for 1 h at 23 °C. The labeled oocytes were mounted and the images were captured using an epifluorescence microscope . Fluorescence intensities were measured using ImageJ software . The distributions of Golgi and ER were classified as below  according to methods described previously [The mitochondria of the oocytes were stained using MitoTracker Green FM . Mature oocytes were blocked with 2% BSA-PBS for 2 h at 37 °C, washed three times in 0.2% PVA-PBS, and stained with 200 nM MitoTracker Green for 30 min at room temperature. After washing three times, the samples were mounted and the images were captured using an epifluorescence microscope . Fluorescence intensities were measured using ImageJ software . The mitochondrial distribution was modified according to the method described by Pawlak et al. . (a) HomGAPDH and RN18S, using the equation R = 2 −∆∆Ct. Primer sequences are shown in Total mRNA was extracted from 100 oocytes in each group using a TRIzol reagent (Invitrogen), according to the manufacturer’s protocol. The complementary DNA (cDNA) was produced using amfiRivert cDNA Synthesis Platinum Master Mix . A PCR reaction mix  was prepared by adding 1 µL of cDNA, 0.4 µL (10 pmol/µL) of forward and reverse primers, 8.2 µL of nuclease-free water , and 10 µL of SYBR Premix Ex Taq , and then amplified on a StepOneTM Real-Time PCR System . The amplification protocol included an initial denaturation step for 10 min at 95 °C, followed by 40 cycles consisting of denaturation for 15 s at 95 °C, annealing for 1 min at 60 °C, and extension for 1 min at 72 °C. The expression of each target gene was quantified relative to the reference genes After 42 h of IVM, mature oocytes from each group were washed three times in PBS and lysed in 60 μL of lysis buffer  containing 1% phenylmethylsulfonyl fluoride  for 3 h. The samples were then boiled for 5 min at 100 °C. Approximately 1 μg of total protein was loaded onto a 12% ExpressPlusTM PAGE Gel . The gel was then transferred onto a nitrocellulose (NC) membrane . Non-specific sites were blocked using Tris-buffered saline plus 0.05% Tween 20 (TBST) overnight in primary antibodies  at 4 °C. The NC membrane was washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibodies, incubated in SuperSignal West Femto Maximum Sensitivity substrate  for 10 min, and then scanned using a Fujifilm LAS-3000 Imaging System . The bands were quantified using the ImageJ version 1.46 r software.p < 0.05 were considered statistically significant.Data are presented as mean ± standard mean of error (SEM). Significant differences were determined using Duncan’s test, following parametric one-way analysis of variance (ANOVA) with the statistical software SPSS . Differences with p < 0.05).In order to evaluate porcine oocyte nuclear maturation, we supplemented the oocytes with different concentrations of TA during IVM. The results showed that there was no significant difference in nuclear maturation among the groups . The WB results showed that p-CDK1 levels were significantly upregulated up to 1.30-fold and 1.36-fold in response to 1 and 10 μg/mL TA, respectively .Additionally, we measured the protein levels of GDF9 and BMP15 in the oocytes. As shown in p < 0.05; RAB3A and ASTL) expression was significantly upregulated in the 1 and 10 μg/mL TA groups , and 100 μg/mL TA (27.84 ± 1.83%) showed a reduction in the normal distribution rate compared to the control  and 67.55 ± 4.86% (10 μg/mL TA) compared with that of the control . Moreover, ER activity and stress were significantly upregulated with increased RCN1 mRNA expression, and downregulated with decreased CHOP mRNA expression upon 1 and 10 μg/mL TA supplementation, respectively . Additionally, anti-polyspermic genes, such as JUNO, CD9, and ZP2, were significantly upregulated with 1 and 10 μg/mL TA supplementation  is accelerated [JUNO, CD9, and ZP2) were upregulated by 1 and 10 μg/mL TA supplementation. Furthermore, CGs and Golgi-related genes  were drastically increased with 1 and 10 μg/mL TA supplementation, resulting in higher rates of normal distribution of CGs and Golgi. Interestingly, the proportion of normal fertilized oocytes (2PN) was improved with 1 and 10 μg/mL TA supplementation by reducing the polyploid (>3PN) rate. Additionally, our results showed that although the cleavage and total cell numbers/blastocyst were not affected, the blastocyst formation rate was significantly increased by improving oocyte quality during IVM media supplementation with 1 and 10 μg/mL TA. Therefore, our results demonstrated that TA supplementation effectively prevents polyspermic fertilization by production, migration, and distribution of CGs, which is critical to cortical reaction and zona hardening, and thereby effectively block the penetration of the second sperm into the oocyte and improve the competence of the embryo development.Another way to prevent polyspermy is the fusion of the first sperm, which causes depolarization of the membrane and shedding of the egg’s sperm receptor Juno . Female elerated . In thisRCN1 expression (inhibiting ER stress-induced apoptosis), and decrease the CHOP expression (inducing ER stress and apoptosis). ER plays several roles in the cell, including protein synthesis and lipid metabolism [2+), thus regulating intracellular Ca2+ homeostasis [Following experiments, we examined the other organelles’ function and distribution, as well as their related gene expressions, including the ER and mitochondria. Our results showed that TA could improve the normal distribution rate of ER, increase the tabolism . ER stretabolism . Therefotabolism . The ER eostasis . ER streeostasis . Moreoveeostasis . TherefoPGC1α, TFAM, POLG, NRF1, and TFB1M) increased with 1 and 10 μg/mL TA supplementation in porcine oocytes. In contrast, severe oxidative stress triggered a decrease in cellular mitochondrial content through the suppression of mitochondrial biogenesis, as well as mitochondrial function [Mitochondrial biogenesis is modulated at the transcriptional, translational, and post-translational levels , and PGCfunction . In concIn the current study, we demonstrated that TA improved the blastocyst formation rate by effectively improving oocyte quality and eliminating the incidence of polyspermic penetration, which is mainly modulated by TA-induced ooplasmic improvement, especially the normal distribution rate of cytoplasmic organelles and their functions and activities. These results provide the basis for the rational development of new fertility treatments.