Multimodal Telemonitoring for Weight Reduction in Patients With Sleep Apnea: A Randomized Controlled Trial.

BACKGROUND: Telemonitoring the use of CPAP devices and remote feedback on device data effectively optimizes CPAP adherence in patients with OSA. RESEARCH QUESTION: Can expanding the scope of telemonitoring and remote feedback to body weight (BW), BP, and physical activity enhance efforts for BW reduction in Patients with OSA receiving CPAP? STUDY DESIGN AND METHODS: Participants were recruited from patients at 16 sleep centers in Japan with OSA and obesity who were receiving CPAP therapy. Obesity was defined as a BMI of ≥ 25 kg/m2, based on Japanese obesity guidelines. Implementation of CPAP telemonitoring was enhanced with electronic scales, BP monitors, and pedometers that could transmit data from devices wirelessly. Participants were randomized to the multimodal telemonitoring group or the usual CPAP telemonitoring group and were followed up for 6 months. Attending physicians provided monthly telephone feedback calls to the usual CPAP telemonitoring group on CPAP data obtained remotely. In the multimodal telemonitoring group, physicians additionally encouraged participants to reduce their BW, after sharing the remotely obtained data on BW, BP, and step count. The primary outcome was set as ≥ 3% BW reduction from baseline. RESULTS: One hundred sixty-eight participants (BMI, 31.7 ± 4.9 kg/m2) completed the study, and ≥ 3% BW reduction occurred in 33 of 84 participants (39.3%) and 21 of 84 participants (25.0%) in the multimodal telemonitoring and usual CPAP telemonitoring groups, respectively (P = .047). Whereas no significant differences were found between the two groups in the change in office and home BP, daily step counts during the study period were significantly higher in the multimodal telemonitoring group than in the usual CPAP telemonitoring group (4,767 steps/d [interquartile range (IQR), 2,864-6,617 steps/d] vs 3,592 steps/d [IQR, 2,117-5,383 steps/d]; P = .02) INTERPRETATION: Multimodal telemonitoring may enhance BW reduction efforts in patients with OSA and obesity. TRIAL REGISTRY: UMIN Clinical Trials Registry; No.: UMIN000033607; URL: www.umin.ac.jp/ctr/index.htm.

A Randomized Controlled Trial of Telemedicine for Long-Term Sleep Apnea Continuous Positive Airway Pressure Management.

Rationale: The effects of telemedicine on adherence in patients with obstructive sleep apnea with long-term continuous positive airway pressure (CPAP) use have never been investigated.Objectives: To examine effects of a telemedicine intervention on adherence in long-term CPAP users.Methods: In a prospective, randomized, multicenter noninferiority trial conducted in 17 sleep centers across Japan, patients who had used CPAP for >3 months and were receiving face-to-face follow-up by physicians every 1 or 2 months were randomized by a coordinating center in a blind manner to the following three groups: 1) follow-up every 3 months accompanied by a monthly telemedicine intervention (telemedicine group: TM-group), 2) follow-up every 3 months (3-month group: 3M-group), or 3) monthly follow-up (1-month group: 1M-group). Each group was followed up for 6 months. The change in percentage of days with ≥4 h/night of CPAP use from baseline to the end of the study period was evaluated. A decline of ≥5% from baseline was considered deterioration of adherence. Noninferiority of TM- and 3M-groups compared with the 1M-group according to the number of patients with deterioration of adherence was evaluated with the Farrington and Manning test (noninferiority margin 15%).Results: A total of 483 patients were analyzed (median duration of CPAP use, 29 [interquartile range, 12-71] mo), and deterioration of adherence was found in 41 of 161 (25.5%), 55 of 166 (33.1%), and 35 of 156 (22.4%) patients in the TM-, 3M-, and 1M-groups, respectively. The noninferiority of the TM-group compared with the 1M-group was verified (difference in percentage of patients with adherence deterioration, 3.0%; 95% confidence interval [CI], -4.8% to 10.9%; P < 0.01). Conversely, the 3M-group did not show noninferiority to the 1M-group (percentage difference, 10.7%; 95% CI, 2.6% to 18.8%; P = 0.19). In the stratified analysis, adherence in TM- and 1M-group patients with poor adherence at baseline improved (TM: 45.8% ± 18.2% to 57.3% ± 24.4%; P < 0.01; 1M: 43.1% ± 18.5% to 53.6% ± 24.3%; P < 0.01), whereas that of the 3M-group did not (39.3% ± 20.8% to 39.8% ± 24.8%; P = 0.84).Conclusions: Intensive telemedicine support could help to optimize CPAP adherence even after long-term CPAP use.Clinical trial registered with www.umin.ac.jp/ctr/index.htm (trial number: UMIN000023118).

EPC1/TIP60-Mediated Histone Acetylation Facilitates Spermiogenesis in Mice.

Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.

Impact of Obstructive Sleep Apnea on Liver Fat Accumulation According to Sex and Visceral Obesity.

RATIONALE: Associations between obstructive sleep apnea (OSA) and liver fat accumulation have been frequently investigated because both morbidities are common. Visceral fat was reported to be closely related to OSA and liver fat accumulation. Recently, sex differences in the association between OSA and mortality have gained much attention. OBJECTIVES: To investigate the associations among OSA, liver fat accumulation as determined by computed tomography, and visceral fat area and their sex differences. METHODS: Studied were 188 males and 62 females who consecutively underwent polysomnography and computed tomography. RESULTS: Although the apnea-hypopnea index was positively correlated with liver fat accumulation in the total males, none of the OSA-related factors was independently associated with liver fat accumulation in either the total male or female participants in the multivariate analyses. When performing subanalyses using a specific definition for Japanese of obesity or visceral obesity (body mass index (BMI) ≥25 kg/m2 or visceral fat area ≥100 cm2), in only males without visceral obesity, percent sleep time with oxygen saturation <90%, in addition to BMI, insulin resistance, and serum triglyceride values, was independently correlated with liver fat accumulation (R2 = 15.1%, P<0.001). In males, percent sleep time of oxygen saturation <90% was also a determining factor for alanine aminotransferase values regardless of visceral fat area. In contrast, OSA was not associated with liver fat accumulation or alanine aminotransferase values in females whether or not visceral obesity was absent. CONCLUSIONS: Sex differences in the visceral fat-dependent impact of OSA on liver fat accumulation existed. Although the mechanisms are not known and ethnic differences may exist in addition to the specific criteria of visceral obesity in Japan, the treatment of male patients with OSA might be favorable from the viewpoint of preventing liver fat accumulation and liver dysfunction even in patients without obvious visceral fat accumulation.

Obesity hypoventilation syndrome in Japan and independent determinants of arterial carbon dioxide levels.

BACKGROUND AND OBJECTIVE: Obesity hypoventilation syndrome (OHS) prevalence was previously estimated at 9% in patients with obstructive sleep apnoea (OSA) in Japan. However, the definition of OSA in that study was based on an apnoea-hypopnoea index (AHI) of ≥ 20/h rather than ≥ 5/h. Therefore, the prevalence of OHS in OSA was not measured in the same way as for Western countries. Our study objectives were to investigate the characteristics of Japanese patients with OHS. METHODS: Nine hundred eighty-one consecutive patients investigated for suspected OSA were enrolled. At least 90% of them were from urban areas, including 162 with obese OSA (body mass index (BMI) ≥ 30 kg/m(2) and AHI ≥ 5/h). RESULTS: The prevalence of OHS (BMI 36.7 ± 4.9 kg/m(2) ) in OSA and that in obese OSA were 2.3% and 12.3%, respectively. Multiple regression analysis revealed that independent of age and BMI, arterial oxygen pressure (contribution rate (R(2) ) = 7.7%), 4% oxygen desaturation index (R(2) = 8.9%), carbon monoxide diffusing capacity/alveolar volume (R(2) = 8.3%), haemoglobin concentration (R(2) = 4.9%) and waist circumference (R(2) = 4.9%) were independently associated with arterial carbon dioxide pressure. After 12.3 ± 4.6 months of CPAP treatment, more than 60% of OHS patients no longer had hypercapnia. CONCLUSIONS: The prevalence of OHS in OSA in Japan was 2.3%. The mean BMI of patients with OHS in Japan was lower than that in Western countries (36.7 kg/m(2) vs 44.0 kg/m(2) ).

Impact of obstructive sleep apnea on abdominal aortic diameters.

Although obesity has been reported to be a potential risk factor for abdominal aortic dilatation, the impact of obstructive sleep apnea (OSA) on the abdominal aortic diameter remains unknown. We retrospectively reviewed 427 patients aged >45 years who underwent polysomnography and abdominal computed tomography from November 2008 to February 2012. Aortic diameters were measured at 3 locations: upper, infrarenal, and lower abdominal aorta. OSA was defined as non-OSA (apnea-hypopnea index [AHI] <10, n = 58), mild to moderate (AHI 10 to 30, n = 167), and severe (AHI ≥30, n = 202). Adjusted diameter was not significantly different among OSA severity categories at the upper (21.0, 21.3, and 21.4 mm, respectively) and infrarenal aorta (19.5, 20.2, and 19.9 mm, respectively) but was significantly different at the lower abdominal aorta (17.3, 18.2, and 18.2 mm, respectively, p = 0.006) with larger diameters in patients with OSA. Multivariate linear regression analyses revealed that risk profiles for aortic dilatation varied according to the location and gender and that OSA (AHI ≥10) was an independent risk factor for infrarenal and lower abdominal aortic dilatation only in men (ß = 0.10 and 0.18, p = 0.049 and 0.001, respectively). In conclusion, OSA may enhance dilatation of the distal abdominal aorta in men.

Differences in associations between visceral fat accumulation and obstructive sleep apnea by sex.

RATIONALE: The difference in mortality from obstructive sleep apnea (OSA) by sex is an important issue. Visceral fat, a significant risk factor for cardiovascular disease, was reported to be closely related to OSA. OBJECTIVES: To assess the different associations between OSA and visceral fat area (VFA) by sex, which might account for the different prognosis in men and women with OSA. METHODS: Participants were 271 men and 100 women consecutively hospitalized for examination of OSA from October 2008 to December 2010. Among the 371 participants, relationships were analyzed between fat areas by computed tomography, comorbidity, polysomnographic data, arterial blood gas, pulmonary function, and venous blood data. Multiple regression analyses were performed to identify variables independently associated with VFA and subcutaneous fat area for each sex. MEASUREMENTS AND MAIN RESULTS: Despite similar body mass index (BMI) and waist circumference, men had larger VFA, more severe OSA, and more severe dyslipidemia than women. Multiple regression analyses revealed that in men, not only age and BMI but also minimal oxygen saturation (contribution rate [R(2)], 4.6%) during sleep, and alveolar-arterial oxygen difference (R(2) = 7.6%) were independently associated with VFA. Conversely, VFA was associated only with BMI in women. CONCLUSIONS: Only in men was OSA independently associated with VFA. The lesser associations between OSA and visceral fat in women might account for the lower impact of OSA on cardiovascular disease or mortality in women.

The additive impact of periodic limb movements during sleep on inflammation in patients with obstructive sleep apnea.

RATIONALE: Both periodic limb movements during sleep (PLMS) and obstructive sleep apnea (OSA) are major causes of sleep disorders and have been associated with systemic inflammation and cardiovascular events. However, it is uncertain whether in combination they promote a higher inflammatory response and greater risk of cardiovascular events than each condition alone. OBJECTIVES: To investigate whether the presence of PLMS is associated with increased inflammation in patients suspected of having OSA. METHODS: In 342 patients who underwent polysomnography to diagnose OSA, plasma C-reactive protein (CRP) and fibrinogen levels were measured. MEASUREMENTS AND MAIN RESULTS: OSA was found in 254 patients, with 46 also having PLMS. Among the 88 patients who did not have OSA, 8 had PLMS. Plasma CRP and fibrinogen levels in the group with both PLMS and OSA were higher than in patients with neither OSA nor PLMS and in patients with OSA only (CRP: 0.20 ± 0.48 vs. 0.09 ± 0.15 vs. 0.13 ± 0.18 mg/dl, P = 0.03; fibrinogen: 298.2 ± 76.1 vs. 269.0 ± 57.1 vs. 270.0 ± 52.6 mg/dl, P < 0.01). Multivariate analysis showed that the presence of PLMS was associated with higher plasma CRP levels (ß = 0.1401, P < 0.01) and fibrinogen levels (ß = 0.1359, P = 0.01) independently from other clinical variables such as body mass index and the severity of OSA. CONCLUSIONS: PLMS were positively associated with plasma CRP and fibrinogen levels in patients suspected of having OSA. Because plasma levels of these proteins have been established as predictive factors of future cardiovascular events, the presence of PLMS may be a useful clinical sign to identify patients with OSA at high risk of cardiovascular events.

Flexible positive airway pressure improves treatment adherence compared with auto-adjusting PAP.

STUDY OBJECTIVES: There are no clinical data comparing adherence and quality of life between auto-adjusting positive airway pressure (APAP) and two different flex positive airway pressure (PAP) devices (A-Flex, C-Flex) in patients with obstructive sleep apnea (OSA). DESIGN AND SETTING: Ninety-three patients in whom OSA was newly diagnosed were randomly assigned to receive 3 mo of APAP (n = 31), APAP with C-Flex (n = 31), or APAP with A-Flex (n = 31). Objective adherence was determined after 3 mo of CPAP treatment, and the Epworth Sleepiness Scale (ESS), Pittsburgh Sleep Quality Index (PSQI), and Calgary Sleep Apnea Quality of Life Index (SAQLI) were examined at baseline and after 3 mo. After 3 mo, patients in the APAP with A-Flex group and those in the APAP with C-Flex group were crossed over and those in the APAP group were switched to A-Flex for an additional 3 mo. MEASUREMENTS AND RESULTS: The groups were similar demographically. Treatment adherence during the first 3 mo was significantly greater in the APAP with C-Flex group (APAP with C-Flex: 5.19 ± 1.84 h/night versus APAP: 3.96 ± 1.66 h/night versus APAP with A-Flex: 4.27 ± 2.12 h/night, P = 0.04). There was a significant improvement in two of four of the SAQLI domain scores and in the ESS and PSQI in the APAP with C-Flex group. Adherence significantly improved among the poor compliers (< 4 h/night of use) in the APAP group after change to APAP with A-Flex (P = 0.01). CONCLUSIONS: Of these three modes of PAP delivery, adherence was greatest with APAP with C-Flex. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00873977.

Association between plasma neutrophil gelatinase associated lipocalin level and obstructive sleep apnea or nocturnal intermittent hypoxia.

BACKGROUND: Both obstructive sleep apnea (OSA) and a novel lipocalin, neutrophil gelatinase associated lipocalin (Ngal), have been reported to be closely linked with cardiovascular disease and loss of kidney function through chronic inflammation. However, the relationship between OSA and Ngal has never been investigated. OBJECTIVES: To evaluate the relationship between Ngal and OSA in clinical practice. METHODS: In 102 patients, polysomnography was performed to diagnose OSA and plasma Ngal levels were measured. The correlations between Ngal levels and OSA severity and other clinical variables were evaluated. Of the 46 patients who began treatment with continuous positive airway pressure (CPAP), Ngal levels were reevaluated after three months of treatment in 25 patients. RESULTS: The Ngal level correlated significantly with OSA severity as determined by the apnea hypopnea index (r = 0.24, p = 0.01) and 4% oxygen desaturation index (ODI) (r = 0.26, p = 0.01). Multiple regression analysis showed that the Ngal level was associated with 4%ODI independently of other clinical variables. Compliance was good in 13 of the 25 patients who used CPAP. Although the OSA (4%ODI: 33.1±16.7 to 1.1±1.9/h, p<0.01) had significantly improved in those with good compliance, the Ngal levels were not significantly changed (60.5±18.1 before CPAP vs 64.2±13.9 ng/ml after CPAP, p = 0.27). CONCLUSIONS: Plasma Ngal levels were positively associated with the severity of OSA. However, the contribution rate of OSA to systemic Ngal secretion was small and changes in Ngal levels appeared to be influenced largely by other confounding factors. Therefore, it does not seem reasonable to use the Ngal level as a specific biomarker of OSA in clinical practice.

Association between sleep apnea, sleep duration, and serum lipid profile in an urban, male, working population in Japan.

BACKGROUND: Dyslipidemia is often comorbid with obstructive sleep apnea (OSA), but few population-based studies have investigated their relationship. Short sleep duration is associated with hypertension and diabetes; however, its association with dyslipidemia is not well known. We investigated relationships among OSA, sleep duration, and the lipid profile in a community-based study. METHODS: We measured the respiratory disturbance index (RDI) and sleep duration by a type 3 portable device and actigraph in 275 men in a Japanese company. Fasting blood parameters were obtained from periodic inspection data. RESULTS: According to Japanese criteria, 143 subjects had dyslipidemia. Percent sleep time of oxygen saturation as measured by pulse oximetry (SpO2) < 90% and prevalence of severe OSA were greater and sleep duration and mean SpO2 during sleep were lower in subjects with dyslipidemia than in those without. Univariate analysis showed that the RDI was positively correlated with serum triglyceride (TG) levels (ρ = 0.20, P < .01), and sleep duration was negatively correlated with serum total cholesterol (TC) levels (γ = -0.13, P = .03) and serum low-density lipoprotein cholesterol levels (γ = -0.12, P = .04). Stepwise multiple regression analysis revealed that TG was correlated with RDI (ß = 0.14, P = .02), BMI (ß = 0.20, P < .01), and alcohol intake (ß = 0.20, P < .01), and that TC was correlated with sleep duration (ß = -0.13, P = .03), age (ß = 0.15, P = .02), and waist/hip ratio (ß = 0.15, P = .02). CONCLUSIONS: Short sleep duration was associated with TC levels and RDI was positively associated with TG levels among working-aged men in an urban Japanese company. Correcting the status of OSA and/or short sleep duration might improve the lipid profile and cardiovascular consequences.

A urine biomarker for severe obstructive sleep apnoea patients: lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase (L-PGDS), which is responsible for the biosynthesis of prostaglandin D2, has been reported to have a close connection with cardiovascular disease and sleep regulation. This study aimed to test the hypothesis that the L-PGDS level is a useful marker to identify patients with obstructive sleep apnoea. 64 subjects were enrolled in this prospective study. Urinary concentrations of L-PGDS were measured in the morning. Measurements were made every 4 h in 25 of the 64 patients. Endothelial function was assessed by the reactive hyperaemia peripheral arterial tone index. Circadian variations in L-PGDS concentrations had a significant time-dependent fluctuation (p = 0.0002). L-PGDS was higher in the subjects with severe obstructive sleep apnoea (median 784.7 ng per mg of creatinine, n = 23) than in control subjects (262.1 ng per mg of creatinine, n = 16; p = 0.004) and in those with moderate obstructive sleep apnoea (371.7 ng per mg of creatinine, n = 25; p = 0.0008). After 2 days of continuous positive airway pressure treatment, L-PGDS concentrations in severe obstructive sleep apnoea subjects (n = 12) decreased significantly (p = 0.02) to levels present in control subjects whereas endothelial function did not change significantly. Morning urinary L-PGDS concentrations had significant correlations with the apnoea/hypopnoea index (R2 = 13.9%) and serum high-density lipoprotein cholesterol (R2 = 6.2%), but not with sleepiness. Urinary L-PGDS might be a moderately useful marker to identify patients with severe obstructive sleep apnoea.

Measurement of dyspnea in patients with obstructive sleep apnea.

PURPOSE: Patients with obstructive sleep apnea (OSA) frequently complain of exertional dyspnea. We aimed to assess its related factors and the significance of its measurement in OSA. METHODS: We evaluated 301 subjects with suspected OSA for dyspnea during activities of daily living using the Medical Research Council (MRC) scale. We analyzed the relationships between MRC grades and various subjective and objective indices. Further, the relationship of disease severity based on the apnea/hypopnea index (AHI) with these indices was examined. Results were compared between those obtained using MRC grades and the AHI. RESULTS: Of 301 subjects, 265 were diagnosed with OSA. Their MRC scores were worse than in non-OSA patients. Among OSA patients, 125 had MRC grade 1 (mild), 121 had MRC grade 2 (moderate), and 19 had MRC grade 3 or more (severe) dyspnea. Various measurements differed significantly between groups categorized according to the MRC scale although determinants between mild and moderate groups and between moderate and severe groups differed. AHI categorizations were not significantly related to patient-reported measurements such as the Medical Outcomes Study 36-item short form, Pittsburgh Sleep Quality Index, and Hospital Anxiety and Depression Scale scores, unlike categorization based on the MRC scale. CONCLUSIONS: Dyspnea is an important outcome in OSA although dyspnea in OSA patients is unrelated to the sleep disorder per se. Measurement of dyspnea in patients with OSA might provide further insights into the health of these patients and clinical manifestations of this disease.

Birth of a healthy infant after intracytoplasmic sperm injection using pentoxifylline-activated sperm from a patient with Kartagener's syndrome.

OBJECTIVE: To describe the delivery of a healthy female infant after intracytoplasmic sperm injection (ICSI) using pentoxifylline-activated sperm from a patient with Kartagener's syndrome. DESIGN: Case report. SETTING: Private assisted reproductive technology clinic in Japan. PATIENT(S): A couple with male factor infertility due to Kartagener's syndrome. INTERVENTION(S): Intracytoplasmic sperm injection using ejaculated sperm activated by pentoxifylline. MAIN OUTCOME MEASURE(S): Semen characteristics, sperm ultrastructure, fertilization, pregnancy, and birth after ICSI. RESULT(S): The fertilization rate was 7 of 12 (58.3%), and the blastocyst formation rate was 4 of 7 (57.1%); all blastocysts were vitrified. After a single blastcyst transfer, a pregnancy ensued and progressed to term; a healthy female infant was delivered. CONCLUSION(S): With ejaculated sperm, which was activated by pentoxifylline, successful fertilization was accomplished by ICSI; thus, fertilization, vitrification, pregnancy, and delivery are attainable with sperm obtained from men with Kartagener's syndrome.

Transplantation of human bone marrow stromal cell-derived Schwann cells reduces cystic cavity and promotes functional recovery after contusion injury of adult rat spinal cord.

The aim of this study was to evaluate whether transplantation of human bone marrow stromal cell-derived Schwann cells (hBMSC-SC) promotes functional recovery after contusive spinal cord injury of adult rats. Human bone marrow stromal cells (hBMSC) were cultured from bone marrow of adult human patients and induced into Schwann cells (hBMSC-SC) in vitro. Schwann cell phenotype was confirmed by immunocytochemistry. Growth factors secreted from hBMSC-SC were detected using cytokine antibody array. Immunosuppressed rats were laminectomized and their spinal cords were contused using NYU impactor (10 g, 25 mm). Nine days after injury, a mixture of Matrigel and hBMSC-SC (hBMSC-SC group) was injected into the lesioned site. Five weeks after transplantation, cresyl-violet staining revealed that the area of cystic cavity was smaller in the hBMSC-SC group than that in the control group. Immunohistochemistry revealed that the number of anti-growth-associated protein-43-positive nerve fibers was significantly larger in the hBMSC-SC group than that in the control group. At the same time, the number of tyrosine hydroxylase- or serotonin-positive fibers was significantly larger at the lesion epicenter and caudal level in the hBMSC-SC group than that in the control group. In electron microscopy, formation of peripheral-type myelin was recognized near the lesion epicenter in the hBMSC-SC group. Hind limb function recovered significantly in the hBMSC-SC group compared with the control group. In conclusion, the functions of hBMSC-SC are comparable to original Schwann cells in rat spinal cord injury models, and are thus potentially useful treatments for patients with spinal cord injury.

Adhesion between plasma membrane and mitochondria with linking filaments in relation to migration of cytoplasmic droplet during epididymal maturation in guinea pig spermatozoa.

High-resolution microscopy has been used to investigate the mechanism of the migration of cytoplasmic droplets during epididymal maturation of guinea pig spermatozoa. On testicular spermatozoa, droplets are located at the neck and, after passage through the middle cauda epididymidis, migrate only as far as the center of the midpiece. Initially, the space between the plasma membrane and outer mitochondrial membranes outside the droplet is 30.8+/-11.0 nm, whereas on mature spermatozoa, it significantly (P<0.01) narrows to a more consistent 15.9+/-1.3 nm. This is accompanied by the appearance of thin filaments cross-linking the two membranes above and below the droplet. Changes also occur in the arrangement of intramembranous particles (IMPs) in the plasma membrane overlying the midpiece. At the spermatid stage, linear arrays of IMPs are absent but appear on immature spermatozoa, where they are short with an irregular orientation, in the epididymis. On mature spermatozoa, numerous parallel linear arrays are present at the region where the plasma membrane adheres to the mitochondria. The membrane adhesion process can thus be observed two-dimensionally. The initial migration of the droplet from the neck is probably attributable to diffusion, with the formation of cross-linking filaments between the two membranes in the proximal midpiece preventing any backward flow and squeezing the droplet distally until it is arrested at the central midpiece by the filaments formed in the distal midpiece. The filaments might also stabilize the flagellum against hypo-osmotic stress encountered during ejaculation and within the female tract.

A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin.

It is important to establish a reliable and progressive model of the acrosome reaction. Here, we present a progression model of the acrosome reaction centering around the acrosomal membrane-anchored protein equatorin (MN9), comparing the staining pattern traced by MN9 antibody immunofluorescence with that traced by Arachis hypogaea agglutinin (PNA)-FITC. Prior to the acrosome reaction, equatorin was present in both the anterior acrosome and the equatorial segment. Since sperm on zona pellucida showed various staining patterns, MN9-immunostaining patterns were classified into four stages: initial, early, advanced, and final. As the acrosome reaction progressed from the initial to the early stage, equatorin spread from the peripheral region of the anterior acrosome toward the center of the equatorial segment, gradually over the entire region of the equatorial segment during the advanced stage, and finally uniformly at the equatorial segment at the final stage. In contrast, the PNA-FITC signals spread more quickly from the peripheral region of the acrosome toward the entire equatorial segment, while decreasing in staining intensity, and finally became weak at the final stage. MN9-immunogold electron microscopy showed equatorin on the hybrid vesicles surrounded by amorphous substances at advanced stage of acrosome reaction. Equatorin decreased in molecular mass from 40-60 to 35 kDa, and the signal intensity of 35 kDa equatorin increased as the acrosome reaction progressed. Thus, the established equatorin-based progression model will be useful for analyzing not only the behavior of equatorin but also of other molecules of interest involved in the acrosome reaction.