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) ).

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.

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.

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.

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).

Equatorin: identification and characterization of the epitope of the MN9 antibody in the mouse.

Equatorin (MN9 antigenic molecule) is a widely distributed acrosomal protein in mammalian sperm. During the acrosome reaction, some amount of equatorin translocates to the plasma membrane, covering the equatorial region. From the results of studies of both in vitro and in vivo fertilization inhibition using the MN9 antibody, equatorin has been suggested to be involved in fusion with the oolemma. In the present study, we cloned equatorin and, using mass spectrometry and carbohydrate staining, found it to be a highly glycosylated protein. Equatorin is a sperm-specific type 1 transmembrane protein, and glycosidase treatment and recombinant protein assays verified that it is an N,O-sialoglycoprotein. In addition, the gamete interaction-related domain recognized by the MN9 antibody is posttranslationally modified. The modified domain was identified near threonine 138, which was most likely to be O-glycosylated when analyzed by amino acid substitution, dephosphorylation, and O-glycosylation inhibitor assays. Immunogold electron microscopy localized the equatorin N-terminus, where the MN9 epitope is present, on the acrosomal membrane facing the acrosomal lumen. These biochemical properties and the localization of equatorin are important for further analysis of the translocation mechanism leading to gamete interaction.

Oocyte activation ability correlates with head flatness and presence of perinuclear theca substance in human and mouse sperm.

BACKGROUND: Recent studies indicate that round-headed sperm cannot activate oocytes and lack the postacrosomal sheath (PAS) or perinuclear theca (PT), although normal flat-headed sperm can activate oocytes and do have PAS (PT). In this study, we investigated how oocyte activation ability correlates with sperm head morphology (round and flat) and the presence of PT, by studying MN13, a representative molecule of the PT. METHODS: We analyzed sperm with flat and round heads from infertile patients with globozoospermia (n = 1) and teratozoospermia (n = 1), and also from GOPC(-/-) mice, an animal model of human globozoospermia. Differential interference contrast image analysis, immunocytochemistry with MN13 antibody, transmission electron microscopy and an oocyte activation assay (assessing pronucleus formation) with ICSI were used. RESULTS: Flat-headed (control) sperm from both a healthy fertile volunteer man and wild-type mice had MN13 and PAS (PT). Flat-headed sperm (<5% of the population) from GOPC(-/-) mice also had both MN13 and PAS (PT), and they showed high oocyte activation ability. In contrast, round-headed sperm from a globozoospermia patient (100%) and GOPC(-/-) mice (>95% of the population) had neither MN13, nor PAS (PT), nor oocyte activation ability. Oocyte activation was higher in flat- versus round-headed sperm from GOPC(-/-) mice (P < 0.05). CONCLUSIONS: Oocyte activation ability may be related to sperm head flatness and presence of MN13 and PAS (PT) in human and mouse sperm. This information is a first step towards the possibility of selecting good-quality sperm with high oocyte activation ability for ICSI.

Oligo-astheno-teratozoospermia in mice lacking RA175/TSLC1/SynCAM/IGSF4A, a cell adhesion molecule in the immunoglobulin superfamily.

RA175/TSLC1/SynCAM/IGSF4A (RA175), a member of the immunoglobulin superfamily with Ca2+-independent homophilic trans-cell adhesion activity, participates in synaptic and epithelial cell junctions. To clarify the biological function of RA175, we disrupted the mouse Igsf4a (Ra175/Tslc1/SynCam/Igsf4a Ra175) gene. Male mice lacking both alleles of Ra175 (Ra175-/-) were infertile and showed oligo-astheno-teratozoospermia; almost no mature motile spermatozoa were found in the epididymis. Heterozygous males and females and homozygous null females were fertile and had no overt developmental defects. RA175 was mainly expressed on the cell junction of spermatocytes, elongating and elongated spermatids (steps 9 to 15) in wild-type testes; the RA175 expression was restricted to the distal site (tail side) but not to the proximal site (head side) in elongated spermatids. In Ra175-/- testes, elongated and mature spermatids (steps 13 to 16) were almost undetectable; round spermatids were morphologically normal, but elongating spermatids (steps 9 to 12) failed to mature further and to translocate to the adluminal surface. The remaining elongating spermatids at improper positions were finally phagocytosed by Sertoli cells. Furthermore, undifferentiated and abnormal spermatids exfoliated into the tubular lumen from adluminal surfaces. Thus, RA175-based cell junction is necessary for retaining elongating spermatids in the invagination of Sertoli cells for their maturation and translocation to the adluminal surface for timely release.

Importance of forkhead transcription factor Fkhl18 for development of testicular vasculature.

Forkhead transcription factors are characterized by a winged helix DNA binding domain, and the members of this family are classified into 20 subclasses by phylogenetic analyses. Fkhl18 is structurally unique, and is classified into FoxS subfamily. We found Fkhl18 expression in periendothelial cells of the developing mouse fetal testis. In an attempt to clarify its function, we generated mice with Fkhl18 gene disruption. Although KO mice developed normally and were fertile in both sexes, we frequently noticed unusual blood accumulation in the fetal testis. Electron microscopic analysis demonstrated frequent gaps, measuring 100-400 nm, in endothelial cells of blood vessels. These gaps probably represented ectopic apoptosis of testicular periendothelial cells, identified by caspase-3 expression, in KO fetuses. No apoptosis of endothelial cells was noted. Fkhl18 suppressed the transcriptional activity of FoxO3a and FoxO4. Considering that Fas ligand gene expression is activated by Foxs, the elevated activity of Foxs in the absence of Fkhl18 probably explains the marked apoptosis of periendothelial cells in Fkhl18 KO mice.

HANP1/H1T2, a novel histone H1-like protein involved in nuclear formation and sperm fertility.

We cloned a testis-specific cDNA from mice that encodes a histone H1-like, haploid germ cell-specific nuclear protein designated HANP1/H1T2. The HANP1/H1T2 protein was specifically localized to the nuclei of murine spermatids during differentiation steps 5 to 13 but not to the nuclei of mature sperm. HANP1/H1T2 contains an arginine-serine-rich domain and an ATP/GTP binding site, and it binds to DNA, ATP, and protamine. To investigate the physiological role of HANP1/H1T2, we generated Hanp1/H1T2-disrupted mutant mice. Homozygous Hanp1/H1T2 mutant males were infertile, but females were fertile. Although a substantial number of sperm were recovered from the epididymides, their shape and function were abnormal. During sperm morphogenesis, the formation of nuclei was disturbed and protamine-1 and -2 were only weakly detectable in the nuclei. The chromatin packaging was aberrant, as demonstrated by electron microscopy and biochemical analysis. The mutant sperm exhibited deficient motility and were not competent to fertilize eggs under in vitro fertilization conditions; however, they were capable of fertilizing eggs via intracytoplasmic sperm injection that resulted in the birth of healthy progeny. Thus, we found that HANP1/H1T2 is essential for nuclear formation in functional spermatozoa and is specifically involved in the replacement of histones with protamines during spermiogenesis. At the time of submission of the manuscript, we found an independent publication by Martianov et al. (I. Martianov, S. Brancorsini, R. Catena, A. Gansmuller, N. Kotaja, M. Parvinen, P. Sassone-Corsi, and I. Davidson, Proc. Natl. Acad. Sci. USA 102:2808-2813, 2005) that reported similar results.

Review of the histological effects of the anti-androgen, flutamide, on mouse testis.

This is a mini-review summarizing recent findings on the effect of flutamide (FLUT), an anti-androgenic toxicant, on the mouse testis, particularly on the ectoplasmic specialization (ES) in the testis. FLUT induces a reduction in the weight of male reproductive tissues, such as the prostate, because it inhibits the formation of the androgen receptors and testosterone retention. The present review summarizes the abnormal histological changes produced in the mouse testis by FLUT. In addition, we outline the effect of FLUT on the expression of cortactin, an actin-binding protein, in the mouse testis. FLUT is often used as a positive control for the identification of endocrine disrupting chemicals having anti-androgenic activities; therefore, a detailed understanding of the adverse effects of FLUT is important for the analysis of the risks to spermatogenesis by anti-androgen-like endocrine disruptors.

Disruption of ectoplasmic specializations between Sertoli cells and maturing spermatids by anti-nectin-2 and anti-nectin-3 antibodies.

AIM: To understand the biological functions of the ectoplasmic specializations between Sertoli cells and maturing spermatids in seminiferous epithelia. METHODS: In order to disrupt the function of the ectoplasmic specializations, nectin-2, which is expressed at the specialization, was neutralized with anti-nectin-2 antibody micro-injected into the lumen of the mouse seminiferous tubule. Anti-nectin-3 antibody was also micro-injected into the lumen in order to neutralize nectin-3, which is expressed at the specialization. RESULTS: The actin filaments at the specialization disappeared, and exfoliation of maturing spermatids was observed by electron microscopy. CONCLUSION: Nectin-2 was neutralized by anti-nectin-2 antibody and nectin-3 was neutralized by anti-nectin-3 antibody, respectively. Inactivated nectin-2 and nectin-3 disrupted the nectin-afadin-actin system, and finally the actin filaments disappeared. As a result, the specialization lost the holding function and detachment of spermatids was observed. One of the functions of the specialization seems to be to hold maturing spermatids until spermiation.

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function.

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

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.

Neonatal administration of diethylstilbestrol has adverse effects on somatic cells rather than germ cells.

Neonatal administration of diethylstilbestrol (DES) to rodents has adverse effects on spermatogenesis. However, not many studies have been conducted to determine which type of cell - germ or somatic - is the major target of DES. In order to clarify this, we tried reciprocal germ cell transplantation--transplantation of germ cells from DES-treated mice into intact mice and germ cells from normal mice into DES-treated mice. The donor germ cells were tagged with the green fluorescent protein (GFP) gene in order to distinguish the exogenous germ cells from the endogenous cells. Moreover, to obtain a large number of spermatogonia from the testes of adult mice, we performed fractionation by centrifugation with Percoll. Consequently, we found that the germ cells collected from DES-treated mice have differentiated into normal sperms in normal seminiferous tubules. However, in the case of the transplantation of normal germ cells into the seminiferous tubules of DES-treated mice, defective spermatogenesis was observed. In conclusion, DES has adverse effects on the somatic cells that are involved in spermatogenesis rather than the germ cells.

Deletion of macrophage migration inhibitory factor gene induces down regulation of sex hormones and ultrastructural abnormalities in mouse testes.

Macrophage migration inhibitory factor (MIF) is a multifunctional protein that acts as a pro-inflammatory cytokine, pituitary hormone, immunoregulator and mitogen. The MIF gene knockout (MIFKO) mouse has been used to study the MIF response in many tissues, such as with skin injury and spinal cord injury; however, there is little information about the MIFKO mouse testis. This study reports the levels of serum and intratesticular estradiol and testosterone, the ultrastructure of the testis, and preliminary findings from in vitro fertilization. Our results revealed a decrease in estradiol and testosterone levels and deformation in spermiogenesis, in the MIFKO mouse. These initial findings study may lead to a better understanding of the role that MIF plays in the mouse testis.

Flutamide depresses expression of cortactin in the ectoplasmic specialization between the Sertoli cells and spermatids in the mouse testis.

Flutamide (FLUT) has potent anti-androgenic activity and is used in the medical field and in a screening test to detect endocrinologically active compounds. Our previous study demonstrated that FLUT induced histological deformation of spermatids and ultrastructural defects of the apical ectoplasmic specialization (ES) in the mouse testis. The apical ES is an actin-based junctional structure between the Sertoli cells and germ cells. Cortactin, an actin-binding protein, is found in the actin layer of ES. The protein level of cortactin was decreased in FLUT-treated testes as shown by Western blot analysis. The detailed analysis indicated that the protein level was drastically decreased in FLUT-treated seminiferous tubules of stages from VI to IX. Immunohistochemistry and immunoelectron microscopy showed that FLUT depressed cortactin expression in the apical ES. In addition, the effect of FLUT on cortactin localization appeared between 12 h and 8 days (about 180 h) after a one-day treatment. These results suggest that FLUT depressed the expression of cortactin in the apical ES with stage specificity. Therefore, the initial target of FLUT may be the cell-cell interactions between the Sertoli and germ cells. To our knowledge, this study is the first to document the decrease of cortactin expression in the abnormal apical ES following treatment with FLUT.