Utility of ultrasonography in a mouse model of non-alcoholic steatohepatitis induced by a choline-deficient, high-fat diet and dextran sulfate sodium.

Background: Nonalcoholic steatohepatitis (NASH) is a chronic progressive liver disease that can progress to cirrhosis and hepatocellular carcinoma. The prevalence of NASH is increasing year by year. However, the etiology and progression of NASH, along with the processes leading to carcinogenesis, remain poorly understood. A range of animal models are used in research, but investigators have been unable to establish a model that results in tumorigenesis from a stable disease state. The present study aimed to create a stable, low-mortality model of NASH using abdominal ultrasonography (US) to assess NASH stage and diagnose liver tumors. Methods: Thirty-four 19-week-old male C57BL/6J mice were fed a choline-deficient, high-fat (CDHF) diet. Twenty animals were given seven courses of 0.8 % dextran sulfate sodium (DSS) for 7 days followed by 10 days of MilliQ water (CDHF+DSS group). The remaining 14 animals drank only MilliQ water (CDHF group). All animals were weighed weekly and US was performed on Days 35 and 120. After necropsy, samples were taken for biochemical analysis and histopathological evaluation. Results: The CDHF+DSS group had significantly lower body weight on Days 35 and 120, and significantly higher liver/body weight (%) on Day 35 compared to the CDHF group. US on Days 35 and 120 revealed significantly shorter long intestine and higher colonic histological score in the CDHF+DSS group compared to the CDHF group. IL-1ß and IL-6 levels in the large intestinal tissue were significantly higher in the CDHF+DSS group. Conclusions: A stable, low-mortality model of NASH was created with a CDHF diet and intermittent 0.8 % DSS. Abdominal US can assess the degree of fatty degeneration and evaluate liver tumorigenesis without necropsy. This assessment procedure will reduce the number of mice killed unnecessarily during experiments, thereby contributing to animal welfare.

Emu Oil and Saireito in combination reduce tumour development and clinical indicators of disease in a mouse model of colitis-associated colorectal cancer.

BACKGROUND: Emu Oil (EO) previously demonstrated therapeutic potential in a mouse model of colitis-associated CRC (CA-CRC). Saireito, a traditional Japanese medicine, has not been investigated in CA-CRC. AIM: To determine whether EO and Saireito could be therapeutic in an azoxymethane (AOM)/dextran sulphate sodium (DSS) model of CA-CRC. METHODS: Female C57BL/6 mice were assigned to groups (n = 10/group); 1) saline control, 2) saline+Saireito, 3) saline+EO, 4) saline+EO/Saireito, 5) AOM/DSS control, 6) AOM/DSS+Saireito, 7) AOM/DSS+EO and 8) AOM/DSS+EO/Saireito. Mice were intraperitoneally injected with saline or AOM (7.4 mg/kg) on day 0 and underwent three DSS/water cycles (2%w/v DSS for 7 days, 14 days water). Mice were orally-gavaged with either water (80 µL), Saireito (80 µL), EO (80 µL) or EO/Saireito (160 µL; 80 µL EO + 80 µL Saireito) thrice weekly. Daily bodyweight and disease activity index (DAI) were recorded and colonoscopies performed on days 20, 41 and 62. Mice were euthanized on day 63. p < 0.05 was considered statistically significant. RESULTS: AOM/DSS induced significant bodyweight loss throughout the trial (max -36%), which was attenuated by Saireito (max +7%), EO (max +5%) and EO/Saireito (max +14%; p < 0.05). AOM/DSS increased DAI compared to saline controls (p < 0.05), which was reduced by Saireito, EO and EO/Saireito (p < 0.05). All treatments reduced colonoscopically-assessed colitis severity (days 20 and 41; p < 0.05). EO/Saireito further decreased colitis severity compared to Saireito and EO alone (day 20; p < 0.05). Finally, EO and EO/Saireito resulted in fewer colonic tumours compared to AOM/DSS controls (p < 0.05). CONCLUSION: Combined EO and Saireito reduced disease and tumour development in AOM/DSS mice, suggesting therapeutic potential in CA-CRC.

Standard protocol devised by the Japanese Pediatric Impedance Working Group for combined multichannel intraluminal impedance-pH measurements in children.

Multichannel intraluminal impedance-pH measurements (MII-pH) are useful for evaluating acid and non-acid gastroesophageal reflux (GER). However, the use of MIH-pH is not yet established in Japan. The Japanese Pediatric Impedance Working Group (Japanese-PIG) convened to devise a standard protocol for MII-pH in Japanese children. The expert members of the Japanese-PIG collected data on pediatric MII-pH from the relevant literature in English, including the standard protocol of MII-pH presented by the European PIG, and the insights of international experts. The resultant consensus was included in the contents of the standard protocol of MII-pH. The standard protocol included standardization of the indication, methodology, and interpretation of MII-pH in Japanese children. The criteria for abnormal GER by MII-pH were defined using the Reflux Index and number of total reflux episodes independently in children aged < 1 year and those aged ≥ 1 year. Moreover, a significant relationship between GER and symptoms was identified using the symptom index and symptom association probability approach. We conclude that the current version of the protocol for MII-pH is tentative because it is not based on data from Japanese children. Further studies are needed to render this protocol clinically beneficial and expand its use in Japan.

Correction to: Standard protocol devised by the Japanese Pediatric Impedance Working Group for combined multichannel intraluminal impedance-pH measurements in children.

The authors would like to add the following sentence in Acknowledgements in the original publication of this paper.

Dye laser treatment for hemorrhagic vascular lesions.

BACKGROUND AND AIMS: It is generally thought that bleeding from a hemangioma is difficult to stop. With development of the long pulse dye laser (LPDL), it has become possible to treat hemangioma with a large blood vessel diameter. Thus, it is effective in treating infantile hemangioma and pyogenic granuloma. MATERIALS AND METHODS: Five patients who visited our hospital from July 2015 to July 2017 due to hemorrhagic hemangioma were treated using a flash lamp excitation pulse dye laser with parameters of 7 mm spot size, 3 msec pulse width, fluence 12-14J/cm2, DCD 30 msec, and delay 30 msec. RESULTS: The bleeding not only stopped, but the raised lesion was flattened in all cases. CONCLUSIONS: LPDL is effective for both infantile hemangioma and pyogenic granuloma. It not only stops bleeding, but also treats the vascular lesions.

Patient confidence regarding secondary lifestyle modification and knowledge of 'heart attack' symptoms following percutaneous revascularisation in Japan: a cross-sectional study.

OBJECTIVE: To assess patient perspectives on secondary lifestyle modification and knowledge of 'heart attack' after percutaneous coronary intervention (PCI) for coronary artery disease (CAD). DESIGN: Observational cross-sectional study. SETTING: A single university-based hospital centre in Japan. PARTICIPANTS: In total, 236 consecutive patients with CAD who underwent PCI completed a questionnaire (age, 67.4±10.1 years; women, 14.8%; elective PCI, 75.4%). The survey questionnaire included questions related to confidence levels about (1) lifestyle modification at the time of discharge and (2) appropriate recognition of heart attack symptoms and reactions to these symptoms on a four-point Likert scale (1=not confident to 4=completely confident). PRIMARY OUTCOME MEASURE: The primary outcome assessed was the patients' confidence level regarding lifestyle modification and the recognition of heart attack symptoms. RESULTS: Overall, patients had a high level of confidence (confident or completely confident,>75%) about smoking cessation, alcohol restriction and medication adherence. However, they had a relatively low level of confidence (<50%) about the maintenance of blood pressure control, healthy diet, body weight and routine exercise (≥3 times/week). After adjustment, male sex (OR 3.61, 95% CI 1.11 to 11.8) and lower educational level (OR 3.25; 95% CI 1.70 to 6.23) were identified as factors associated with lower confidence levels. In terms of confidence in the recognition of heart attack, almost all respondents answered 'yes' to the item 'I should go to the hospital as soon as possible when I have a heart attack'; however, only 28% of the responders were confident in their ability to distinguish between heart attack symptoms and other conditions. CONCLUSIONS: There were substantial disparities in the confidence levels associated with lifestyle modification and recognition/response to heart attack. These gaps need to be studied further and disseminated to improve cardiovascular care.

Surgical exploration for impalpable testis: Which should be first, inguinal exploration or laparoscopic abdominal exploration?

PURPOSE: To discuss an optimal surgical approach for impalpable testis in children, our own treatment results and those reported in the literature were reviewed. MATERIALS AND METHODS: Seventy-two impalpable testes were diagnosed in 68 patients: unilateral in 64 patients and bilateral in 4 patients. All patients underwent surgical exploration at the ages of 6 to 140months (median, 15months). The inguinal canal was initially explored, and abdominal exploration was performed with laparoscopy when an extra-abdominal testis was not identified. In addition, articles regarding surgical exploration for impalpable testis, published over the last 20years, were retrieved and the results were examined. RESULTS: Testes were detected by inguinal exploration in 28 of 72 (39%) impalpable testes: intracanalicular in 22 testes and at the internal inguinal ring (peeping or low abdominal testis) in 6 testes. All these testes were treated by conventional inguinal orchidopexy. Laparoscopic exploration was performed in 44 (61%) impalpable testes, and 4 (5.6%) high abdominal testes were detected and treated by two-stage Fowler-Stephens orchidopexy. Vanishing or absent testis was the final diagnosis in the remaining 40 testes (55.6%). The literature review showed that the ratios of intra- and extra-abdominal testes were lower in the articles that reported the results of inguinal or scrotal exploration than in those of laparoscopic exploration, although the difference was not significant. CONCLUSIONS: Considering the relatively low incidence of high abdominal testis, we recommend to start with inguinal exploration for impalpable testis. When an extra-abdominal testis is not detected, transinguinal laparoscopic exploration should be indicated. LEVEL OF EVIDENCE: Treatment study, Level IV.

Safety and efficacy of selective sac extraction method of inguinal hernia repair in children: results of a prospective study.

PURPOSE: A prospective study was conducted to confirm the safety and efficacy of the selective sac extraction method (SSEM) of inguinal hernia repairs in children. METHODS: Primary endpoints of the study were the incidence of any complication related to the SSEM, or hernia recurrence. Secondary endpoints included the success rate of the SSEM, length of incision at the end of operation, and duration of operation. The incidence of contralateral manifestation of hernia was also examined. RESULTS: Between October 2009 and December 2011, a total of 317 repairs, 145 male repairs and 172 female repairs, were performed by applying the SSEM. There were three operative conversions, and the success rate of the SSEM was 99% in both male and female patients. The length of incision ranged from 4.0 to 12.5 mm (median 6.0 mm) and was ≤7.0 mm in 93% repairs. The incisional length for male repairs ranged from 4.0 to 12.5 mm (median 6.0 mm) and was ≤7.0 mm in 86% repairs, while it ranged from 4.0 to 9.0 mm (median 5.5 mm) in female repairs and was ≤6.5 mm in 96% repairs. The duration of the operation for unilateral repair ranged from 9 to 66 min (median 21 min). Eighty percent of repairs were examined 6-44 months (median 12 months) after the operation. There was one (0.4%) recurrence among 250 repairs and two (1.7%) cases of testicular dislocation among 115 male repairs. Contralateral hernia presented in 19 (9.5%) of 199 patients with unilateral hernia who underwent the follow-up. CONCLUSIONS: The feasibility of the SSEM was reconfirmed, and it was revealed that the complication and recurrence rates were low and acceptable. The SSEM is safe and effective, and should be a standard method for repairing inguinal hernia in children.

Association of extrahepatic bile duct duplication with pancreaticobiliary maljunction and congenital biliary dilatation in children: a case report and literature review.

We herein report a case of cystic-type congenital biliary dilatation (CBD) in whom an extremely rare anomalous duplication of the common bile duct and pancreaticobiliary maljunction were diagnosed intraoperatively by meticulous surgical manipulations via conventional open surgery. By performing a dissection at the outer epicholedochal layer of the cyst, a thin cord-like structure shown to be the distal part of the common bile duct was identified. A further exploration revealed that the most distal (extra- and intrapancreatic) part of the common bile duct was duplicated, and each branch of the duct was connected to the main and accessory pancreatic ducts. The experience with our case and a literature review showed that extrahepatic bile duct duplication is generally associated with pancreaticobiliary maljunction and CBD. We conclude that an extremely careful exploration with delicate and meticulous surgical manipulation is essential to identify these morphological anomalies and prevent intraoperative and postoperative complications of CBD, such as pancreatic duct injury or pancreatitis.

[Diagnosis and treatment of acute appendicitis in children].

In this review article, we discussed the pathogenesis, pathophysiology, diagnosis and treatment of acute appendicitis in children. Indications for early surgery, the operative methods of laparoscopic appendectomy and the treatment outcome are also presented.

A selective sac extraction method: another minimally invasive procedure for inguinal hernia repair in children: a technical innovation with satisfactory surgical and cosmetic results.

PURPOSE: To achieve satisfactory surgical and cosmetic results with minimal surgical invasiveness without laparoscopic assistance in childhood inguinal hernia repair, a novel technique, the selective sac extraction method (SSEM), was devised. The technical feasibility of this method was retrospectively examined. METHODS: The method was applied to 162 consecutive hernia repairs in 153 patients, 79 boys and 74 girls, excluding patients with incarcerated or irreducible hernia, sliding hernia of the ovary (hernia with palpable ovary at repair), or hernia associated with an undescended testis. Hernia repair was performed by several young surgeons under the guidance of one teaching surgeon. RESULTS: Patients' ages ranged from 1 month to 12 years (median, 3 years), and their body weights ranged from 2.9 to 41 kg (median, 14.4 kg). The SSEM was accomplished in 149 (92%) of 162 repairs, and lengths of incision at the end of the procedure were less than 10 mm in all repairs. The success rate was 72 (88%) of 82 repairs in male patients and 77 (96%) of 80 repairs in female patients. In the remaining 13 (8%) repairs in 13 patients, 10 males and 3 females, the procedure was converted to a conventional open method by extending the incision to 10 mm or longer. The ages of the 13 patients ranged from 1 month to 8 years (median, 3 months), being significantly younger than patients whose repair was accomplished by the SSEM (P = .006). The reason for conversion in 9 repairs was a huge or thickened sac that could not be extracted from the small incision, of which 6 involved male infants younger than 11 months. Obesity (the body mass index was 22.2 and >97th percentile for the patient's age) and thick subcutaneous tissue characteristic of early infancy were also reasons, both of which hindered the approach to the inguinal canal. Other reasons included malpositioning of the skin incision and difficulty in sac identification. The lengths of incision at the end of the 162 repairs ranged from 4.5 to 13.0 mm (median, 7.0 mm). Those in female patients, 4.5 to 11.5 mm (median, 6.5 mm), were significantly shorter than in male patients, 5.0 to 13.0 mm (median, 7.5 mm) (P < .001). The median length of the operation was 25 minutes. A follow-up interview revealed that there was no hernia recurrence with a median follow-up time of 20 months (range, 12-29 months). No postoperative complications were reported by the parents. As for the parents' satisfaction with wound cosmesis, 93% of them rated it as good or excellent. CONCLUSIONS: Inguinal hernia repair with the SSEM through a minimal skin incision is technically feasible, with very satisfactory surgical and cosmetic results. As it has the potential to be a standard method for inguinal hernia repair in children, a prospective study to confirm the safety of the procedure should be carried out.

Efficacy of granulocyte apheresis in pediatric patients with ulcerative colitis: a pilot study.

OBJECTIVES: Granulocyte apheresis (GCAP), involving the removal of granulocytes from the blood, may improve clinical symptoms and facilitate a reduction in the dose of steroids in adult patients with ulcerative colitis. As a preliminary trial, GCAP was used to taper the dose of steroids in 4 pediatric patients with ulcerative colitis. METHODS: Three males and 1 female ranging from 11 to 17 years old were treated with GCAP once per week for 5 consecutive weeks/course. The ages of patients at clinical onset ranged from 8 to 12 years and the length of time from the clinical onset to GCAP treatment ranged from 28 to 58 months (median, 38.5 months). RESULTS: In 2 patients, symptoms and signs indicating disease activity improved after 2 courses of GCAP. Laboratory data and endoscopic findings also improved after treatment and the clinical efficacy was judged to be excellent in these patients. In 1 patient, GCAP improved laboratory and endoscopic hallmarks, but bloody stools persisted. Finally, the treatment was ineffective in the fourth patient who eventually underwent surgery. CONCLUSIONS: GCAP is effective in improving clinical symptoms and may play an important role in converting steroid therapy to other treatments in children with steroid-refractory or steroid-dependent ulcerative colitis.

Laparoscopic versus open appendectomy in children with uncomplicated and complicated appendicitis.

PURPOSE: To address whether laparoscopic appendectomy could be an alternative to conventional open appendectomy in children with complicated appendicitis as well as uncomplicated appendicitis, a retrospective study comparing laparoscopic and open appendectomies was performed. METHODS: One hundred patients who were treated by immediate appendectomy, either laparoscopically or by the open method, between May 2000 and August 2003 were included in the study. There were 53 patients in the laparoscopic appendectomy group and 47 patients in the open appendectomy group. RESULTS: The operating time was significantly longer for laparoscopic appendectomy than for open appendectomy (P < .001). The length of hospital stay was significantly shorter in laparoscopic appendectomy in patients with uncomplicated appendicitis (P = .001). Thirteen of the 100 patients (13.0%) had 15 postoperative complications including wound infection (n = 8), intraabdominal abscess (n = 4), stitch abscess (n = 2), and small bowel obstruction (n = 1). In both uncomplicated and complicated appendicitis, there was no significant difference between laparoscopic and open appendectomies in the complication rates, and the incidences of each complication did not differ between the procedures. Among the 14 patients with generalized peritonitis, postoperative complications were seen in 5 patients (35.7%). Although the presence of generalized peritonitis was associated significantly with postoperative complications (P = .017), there was no significant association between the procedure and complications. Overall treatment costs were increased by 26.0% in laparoscopic appendectomy. CONCLUSIONS: Laparoscopic appendectomy should remain an option in children with uncomplicated and complicated appendicitis, and when laparoscopy is selected, consideration of the advantages and disadvantages of the procedure is essential.

Self/nonself recognition in ascidian fertilization: vitelline coat protein HrVC70 is a candidate allorecognition molecule.

Ascidians are hermaphrodites releasing sperm and eggs simultaneously, but many species are self-sterile because of a self/nonself-recognition system in spermegg interaction. Here, we show that a 70-kDa vitelline coat protein, HrVC70, consisting of 12 epidermal growth factor-like repeats, plays a key role in self/nonself recognition during ascidian fertilization. We discovered that the amount of HrVC70 of the self-sterile mature oocytes is markedly higher than that of the self-fertile immature oocytes and that the selfsterile mature oocytes become self-fertile by acid treatment, which is able to release the HrVC70 from isolated vitelline coats. In addition, fertilization is strongly inhibited by the pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm. A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level, with no identical sequences among the 10 individuals tested. Furthermore, genomic DNA analysis revealed that the epidermal growth factor-like domains correspond to the exons, and each intron is highly conserved among even- and odd-numbered introns, suggesting that multiple gene duplications or amplification of this region might have taken place during evolution. It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism probably elicited by crossing over and specific nucleotide substitutions. These results indicate that HrVC70 is a candidate allogeneic recognition molecule in the gamete interaction of the ascidian Halocynthia roretzi.

Infantile intestinal leiomyosarcoma is prognostically favorable despite histologic aggressiveness: case report and literature review.

Intestinal leiomyosarcomas in children are extremely rare. The authors present a case of infantile intestinal leiomyosarcoma of the colon that was resected and was classified histologically as grade 2 because of abundant mitoses. The tumor cells infiltrated the submucosa and the serosal surface. Immunohistochemically, these cells were positive for alpha-smooth muscle actin but not for CD34 or CD117, by which the tumor was differentiated from gastrointestinal stromal tumor (GIST). The patient underwent follow-up without adjuvant therapy and is now doing well with no evidence of the disease for 42 months. Based on the current case and a review of 18 reported cases, it is concluded that infantile intestinal leiomyosarcoma, which is differentiated from GIST, has a favorable prognosis even when the histologic appearance is suggestive of aggressive biological characteristics. Adjuvant therapy is unnecessary when tumors are completely resected.

D-lactic acidosis in short-bowel syndrome managed with antibiotics and probiotics.

D-lactic acidosis sometimes occurs in malabsorbed patients with short-bowel syndrome and is characterized by recurrent episodes of encephalopathy and metabolic acidosis. The characteristic neurologic abnormalities and the presence of metabolic acidosis raises a diagnostic suspicion, and the diagnosis is made when the serum level of D-lactic acid is greater than 3 mmol/L. Standard treatment consists of restricting oral carbohydrates or fasting, correction of metabolic acidosis, and a long-term suppression of pathogenic floras with antibiotics. The authors present a case of D-lactic acidosis in a 22-year-old patient with short-bowel syndrome, to whom intestinal bacterial agents (probiotics) were given in addition to oral kanamycin. Recolonization of the intestine with nonpathogenic floras should be a long-term treatment for D-lactic acidosis.

Umbilicoplasty for large protruding umbilicus accompanying umbilical hernia: a simple and effective technique.

A large umbilical protrusion with redundant skin accompanying an umbilical hernia sometimes needs umbilicoplasty. Although several different techniques for making umbilical depression have been used, the results of the plastic surgery are sometimes unsatisfactory due to postoperative flattening or disappearance of the umbilical depression. To make a permanent umbilical depression that is cosmetically acceptable, we have modified the techniques. Umbilicoplasty was performed in 14 children whose ages ranged from 6 months-6 years and 3 months (median, 1 year and 10 months) and who had umbilical hernia with a large umbilical protrusion. After the fascial defect was closed, the diameter of the umbilicus was reduced to half that before surgery by removing fan-shaped skin flaps and approximating skin edges, and then inverting the umbilicus and fixing it caudally to the fascia and skin. There were no postoperative complications, and no flattening or disappearance of umbilical depression was observed during the follow-up of 10-19 months. The authors' technique of umbilicoplasty for a large protruding umbilicus accompanying umbilical hernia is a simple method that produces acceptable cosmetic results.

Epidermoid cyst: rare testicular tumor in children.

Epidermoid cyst of the testis, extremely rare in children, is a non-teratomatous benign tumor, and testis-sparing surgery should be the treatment of choice. To prevent unnecessary orchiectomy, recognition of this rare tumor in children is essential.

Activation of the aryl hydrocarbon receptor by some vegetable constituents determined using in vitro reporter gene assay.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological action of many aromatic environmental pollutants. In this study, we investigated the activation of the AhR by some vegetable constituents using the AhR-based bioassay for dioxins, i.e., the chemical activated luciferase gene expression (CALUX) assay. Ninety-five vegetable constituents, including flavonoids, tannins, saponins, and terpenes, were tested in vitro. Among them, isoflavones such as daidzein, resveratrol having a stilbene structure, and some flavonoids such as naringenin, hesperetin, and baicalein showed AhR activation.

Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi.

We previously reported that sperm proteasome is responsible for degradation of the ubiquitinated vitelline-coat during fertilization in the ascidian Halocynthia roretzi. Here, we report the roles in fertilization and localization in the sperm cell surface of H. roretzi sperm proteasome. An anti-proteasome antibody, as well as the proteasome inhibitors MG115 and MG132, inhibited the fertilization, indicating that the sperm proteasome functions extracellularly in ascidian fertilization. In order to further assess this issue, the sperm surface proteasome activity was labeled with a cell-impermeable labeling reagent, NHS-LC-biotin, extracted with 0.1% CHAPS, and was subjected to a pull-down assay with avidin-agarose beads. It was found that a substantial amount of sperm proteasome is exposed to the cell surface. Partition analysis with Triton X-114 also revealed that a considerable amount of the sperm proteasome activity is partitioned into a lipid layer. Localization of the proteasome activity was investigated by fluorescence microscopy with succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide as a substrate. The sperm proteasome activity was specifically detected in the sperm head region, and it was markedly activated upon sperm activation. The membrane-associated proteasome was purified from the membrane fraction of H. roretzi sperm by affinity chromatography using an anti-20S proteasome antibody-immobilized Sepharose column. SDS-PAGE of the purified preparation showed a similar pattern of subunit composition to that of the 26S proteasome of mammalian origin. Taken together, these results indicate that H. roretzi sperm has the membrane-associated proteasome on its head, which is activated upon sperm activation, and that sperm proteasome plays an essential role in H. roretzi fertilization.