Complementary and Alternative Medicines Used by Middle-Aged to Older Taiwanese Adults to Cope with Stress during the COVID-19 Pandemic: A Cross-Sectional Survey.

Background: This study aimed to investigate the factors influencing the use of complementary and alternative medicines (CAMs) to manage stress during the COVID-19 pandemic in Taiwan. Methods: A cross-sectional survey was administered to community-dwelling adults between the ages of 46 and 75 years, and a total of 351 participants completed the questionnaire. Log-binominal regression analyses were fitted to explore the factors associated with the use of CAMs. Results: The mean age of the participants was 57.0 years, and 67.0% reported that they had used CAMs within the past three months. Middle-aged adults were more likely to use CAMs than late middle-aged adults and older adults (p < 0.001). Overall, the major CAMs utilized to relieve psychological stress were music therapies (37.6%), massage (31.1%), spinal manipulation (25.1%), relaxing therapies (24.2%), and reading scriptures or The Bible (23.9%). Religion and vegetarian diets were the most important factors influencing participants to use CAMs, especially music therapies, massage, and reading scriptures/The Bible. Conclusions: CAM use was very prevalent among middle-aged adults in Taiwan; in particular, music therapies were the most favored activities for reducing stress. Population-specific mental health interventions using music can be developed to improve stress management outcomes during public health emergencies.

Insulin ameliorates hypoxia-induced autophagy, endoplasmic reticular stress and apoptosis of myocardial cells: In vitro and ex vivo models.

Whether and how insulin counteracts the cytotoxic effects of hypoxia and improves cardiomyocyte viability remains unclear. To achieve this aim, cultured neonatal rat cardiomyocytes pretreated with vehicle or 1 µM insulin were exposed to either normoxic or hypoxia environment for up to 24 h. Cell viability was monitored and cellular apoptosis as well as necrosis, indexes of autophagy, endoplasmic reticular (ER) stress, and expressions of specific relevant mediators of the signaling pathways of autophagy were also assessed. Hypoxia impaired cell viability, induced autophagy, triggered apoptosis, activated ER stress pathway-associated apoptotic responses along with downstream pro-apoptotic transcriptional factor C/EBP homologous protein (CHOP), and increased apoptosis of myocardial cells. On the other hand, insulin pretreatment effectively ameliorated autophagy via PI3-K/Akt signaling pathway, suppressed ER stress, and prevented hypoxia-induced cellular apoptosis. In an ex vivo study, isolated rat hearts were pre-treated in some cases with insulin and subjected to proximal left coronary artery ligation to induce acute myocardial ischemia. Coronary ligation-induced acute ischemia upregulated glucose-related protein 78 (GRP78) and triggered cellular apoptosis in the jeopardized myocardium. Conversely, insulin pretreatment suppressed these hypoxia-related cytotoxic events and reduced myocardial infarct size by up to 15.2%. In conclusion, hypoxia impedes cell viability through triggering autophagy, ER stress and apoptosis, whereas insulin pretreatment effectively prevents these cytotoxic actions of hypoxia, preserves myocardial cell viability and reduces myocardial infarct size. These results indicated the cytoprotective mechanism of insulin against the insult of hypoxia may justify insulin as a therapeutic option for patients with acute myocardial infarction.

Pathobiological Mechanisms of Endothelial Dysfunction Induced by tert-Butyl Hydroperoxide via Apoptosis, Necrosis and Senescence in a Rat Model.

Background: Endothelial dysfunction is one of the underlying causes for vascular diseases. tert-Butyl hydroperoxide (t-BHP), a short-chain lipid hydroperoxide analog, has been reported to cause adverse effects in different systems. However, the adverse actions of t-BHP on inducing endothelial dysfunction are unclear and remain under investigation. Aim of the present study was to identify the pathobiological mechanisms of t-BHP in rat aortic endothelial cells and thoracic aorta. Methods: Primary cultured cells were treated with vehicle or t-BHP (50, 100, 250, 500, and 1,000 µM). Cells were harvested and specific analyses regarding cellular apoptosis, necrosis, and senescence were conducted. Additionally, t-BHP (0.1, 0.2, and 0.4 mmol/kg body weight) or vehicle were administered to male rats (the young group at 6 weeks of age and the mature adult group at 24 weeks of age) daily through intraperitoneal injections. At 10 days after the first drug treatment apoptotic endothelial toxicity was evaluated by biochemical, histological, and immunofluorescent staining analyses. Results: Dose-dependent effects of t-BHP were observed for the reduction of cell viability, deterioration of cell toxicity, initiation of cell cycle arrest, and triggering of apoptosis and necrosis. Moreover, increase of cells stained positive for senescence-associated beta-galactosidase (SA-ß-Gal), amelioration of telomerase activity, and precipitations of necrotic, cell cycle, and apoptotic signaling regulatory proteins were also found in the in vitro model. In the in vivo study, results indicated that t-BHP at higher doses enlarged the intima-medial thickness of descending aorta in the mature adult group, but led to aortic narrowing in the young group. Increased injuries were observed by upregulating endothelial apoptosis- and senescence-positive staining, along with caspase-3 activity and down-regulating telomerase activity. Conclusion: These results confirmed that t-BHP impaired aortic endothelial cell survival at least partially by the activation of p53-mediated signaling pathways, inhibition of cell cycle regulatory proteins, and initiation of cellular senescence-related signaling pathways. In conclusion, t-BHP was found to be a major trigger for impairing aortic endothelial cell survival and deteriorating vascular dysfunction in experimental practice.

Coloring Activities for Anxiety Reduction and Mood Improvement in Taiwanese Community-Dwelling Older Adults: A Randomized Controlled Study.

To investigate the effect of mandala coloring, plaid pattern coloring, and free-form drawing activities on anxiety and mood in older Taiwanese adults. A total of 120 older adults aged 55 years to 75 years were recruited from 18 community-based learning centers for older adults in southern Taiwan. They were randomly assigned to engage in one of the following four activities for 20 minutes: (1) mandala coloring group, (2) plaid pattern coloring group, (3) free-form drawing group, and (4) reading group (control). Information on sociodemographic, lifestyle, and perceived health status was collected at the baseline. In addition, anxiety levels, measured using the 20-item State-Trait Anxiety Inventory-State Anxiety Scale (STAI-S), were ascertained at the baseline (T1), after a brief anxiety induction (T2), and at the end of the assigned activity (T3). The mean anxiety levels among the four groups at T3 were analyzed using analysis of covariance, followed by Sidák multiple comparison test, as appropriate. The mean age of the 120 study participants was 65.1 years and 73.3% were females. A significantly lower anxiety level was observed only in the mandala coloring group (least square mean = 28.2; 95% confidence interval = 24.7-31.7) compared with the control group (least square mean = 36.0; 95% confidence interval = 32.9-39.2) (P=0.004, partial eta-squared = 0.113). Furthermore, when the STAI-S was analyzed at the item level, the mandala coloring group was significantly different from the control group in the following six feelings: calmed down, safe, at ease, rested, satisfied, and I feel good. In conclusion, short-term mandala coloring activity could significantly alleviate self-induced anxiety in community-dwelling older adults. Further studies on the long-term effects of mandala coloring activity in improving the emotional well-being of older adults are warranted.

Dose-response relationship of specific allergen exposure-induced immunological tolerance: a mouse model.

BACKGROUND: It is believed that adequate allergen preimmunization exposure could induce immunologic tolerance. The purpose of this study was to investigate the dose-dependent mechanisms related to antigen-specific tolerance induction in a mouse model. METHODS: Mice were assigned to 5 groups: the control (Cont) group received phosphate-buffered saline (PBS) preimmunization exposure and PBS sham immunization; the other 4 groups were exposed preimmunization to PBS (PBS group) or ovalbumin (OVA) (first mucosal doses: 1.25%, 2.5%, or 5% wt/vol aerosol from days -3 to -1) prior to OVA immunization. The OVA-immunized mice received intraperitoneal doses of 20 µg OVA (on days 1, 7, and 14), and then a second set of mucosal doses with 0.5% wt/vol OVA aerosol (on days 18 to 20). After assessment of airway hyperresponsiveness (AHR), the mice were euthanized and their blood, bronchoalveolar lavage fluids (BALFs), and lung tissues were collected for further analyses. RESULTS: OVA-immunized mice exposed to OVA preimmunization had reduced AHR and immunoglobulin E production when compared to the PBS group. OVA preimmunization exposure inhibited eosinophilic inflammation in lung tissues. The proportions of BALF eosinophil counts from the groups exposed to OVA preimmunization were significantly decreased when compared with those exposed to PBS preimmunization. The balance of T helper 2 (Th2) and T regulatory (Treg) cytokines in BALFs were additionally observed in this mouse model. CONCLUSION: Our results suggest that preimmunization exposure to an appropriate dose of a specific antigen could suppress allergic airway inflammation by induction of immunological tolerance.

Shikonin Induces Apoptosis, Necrosis, and Premature Senescence of Human A549 Lung Cancer Cells through Upregulation of p53 Expression.

Shikonin, a natural naphthoquinone pigment isolated from Lithospermum erythrorhizon, has been reported to suppress growth of various cancer cells. This study was aimed to investigate whether this chemical could also inhibit cell growth of lung cancer cells and, if so, works via what molecular mechanism. To fulfill this, A549 lung cancer cells were treated with shikonin and then subjected to microscopic, biochemical, flow cytometric, and molecular analyses. Compared with the controls, shikonin significantly induced cell apoptosis and reduced proliferation in a dose-dependent manner. Specially, lower concentrations of shikonin (1-2.5 µg/mL) cause viability reduction; apoptosis and cellular senescence induction is associated with upregulated expressions of cell cycle- and apoptotic signaling-regulatory proteins, while higher concentrations (5-10 µg/mL) precipitate both apoptosis and necrosis. Treatment of cells with pifithrin-α, a specific inhibitor of p53, suppressed shikonin-induced apoptosis and premature senescence, suggesting the role of p53 in mediating the actions of shikonin on regulation of lung cancer cell proliferation. These results indicate the potential and dose-related cytotoxic actions of shikonin on A549 lung cancer cells via p53-mediated cell fate pathways and raise shikonin a promising adjuvant chemotherapeutic agent for treatment of lung cancer in clinical practice.

Association between Disease-Specific Quality of Life and Complementary Medicine Use in Patients with Rhinitis in Taiwan: A Cross-Sectional Survey Study.

Rhinitis is a common medical condition and can seriously impact patients' quality of life. The objective of this study was to investigate the association between disease-specific quality of life and use of complementary and alternative medicine (CAM) modalities among Taiwanese rhinitis patients. A cross-sectional survey was undertaken at the outpatient department of otolaryngology in a medical center in Taiwan. Sociodemographic information, disease-specific quality of life (Chinese version of the 31-item Rhinosinusitis Outcome Measure, CRSOM-31), and previous use of CAM modalities for treatment of rhinitis of the patients were ascertained. Factor analysis was performed to reduce the number of CAM modalities. The resulting factors were analyzed for their association with CRSOM-31 score using linear regression analyses. Results from the multiple linear regression analyses indicated that Factor 1 (traditional Chinese medicine), Factor 2 (mind-body modalities), Factor 3 (manipulative-based modalities), female sex, and smoking were significantly associated with a worse disease-specific quality of life. In conclusion, various CAM modalities, female sex, and smoking were independent predictors of a worse disease-specific quality of life in Taiwanese patients with rhinitis.

Factors Associated with the Use of Different Treatment Modalities among Patients with Upper Airway Diseases in Taiwan: A Cross-Sectional Survey Study.

Rhinitis is a common upper airway disease and can have great impact on patients' quality of life. Factors associated with the use of common treatment modalities among 279 Taiwanese rhinitis patients from the outpatient department of otolaryngology in a medical center were investigated using a cross-sectional survey study. Results from multiple logistic regression analysis, adjusted for etiologies of rhinitis, revealed that males were associated with surgical intervention (OR = 2.11, P = 0.009). Lower educational level was associated with oral (OR = 2.31, P = 0.024) and topical medications (OR = 2.50, P = 0.005). Poor or fair general health status was associated with topical medications (OR = 4.47, P = 0.001), whereas very good or excellent general health status was inversely associated with surgical intervention (OR = 0.32, P = 0.002). Smoking was associated with the use of nasal irrigation (OR = 2.72, P = 0.003). Worse disease-specific quality of life was associated with oral medications (OR = 2.46, P = 0.010) and traditional Chinese medicine (OR = 5.43, P < 0.001). In conclusion, the use of different treatment modalities for rhinitis was associated with different combinations of independent factors.

Upper airway inflammation exacerbates bronchial hyperreactivity in mouse models of rhinosinusitis and allergic asthma.

BACKGROUND: Recent studies have suggested that upper airway inflammation has a strong impact on lower airway diseases. The purpose of this study was to assess whether nasal inflammation could exacerbate allergic asthma in a mouse model. METHODS: Mice were assigned to 4 groups: control (Cont), either rhinosinusitis (R) or allergic asthma (A) alone, and both rhinosinusitis and allergic asthma (R&A). Mice underwent induction of nasal inflammation (R and R&A) or sham surgery (Cont and A) on day 1. Mice in the A and R&A groups were sensitized to ovalbumin on days 1, 7, and 14, followed by aerosol challenge on days 18 to 20, whereas in the Cont and R groups only saline was administered. All mice were assessed for airway hyperresponsiveness (AHR) and were euthanized on day 21. The sera, bronchoalveolar lavage fluids (BALFs), and nasal and lung tissues were collected for further analyses. RESULTS: Histology findings confirmed upper and lower airway inflammation in experimental mice. Significantly increased AHR and total serum immunoglobulin E (IgE) were observed in the R&A group when compared with those of the Cont, R, and A groups. Responses to IgG2a induction were also found in sera and BALFs from mice with rhinosinusitis (R and R&A). Higher levels of interleukin 4 (IL-4) and IL-13, and increased eosinophilic inflammation were detected in BALFs and lung tissues from the experimental groups when compared with those from the Cont group. CONCLUSION: Our results confirm that upper airway inflammation could exacerbate allergic asthma, and provide support to the concept of "one airway, one disease.

[Factors associated with self-care behaviors in middle-aged adults and elderly with diabetes mellitus].

BACKGROUND: Adequate self-care behavior in diabetic individuals is essential to reduce the progress of the condition and reduce diabetes related complications. Continued research to understand the factors associated with self-care behaviors is needed in order to reduce health care costs and the social burdens associated with diabetes. PURPOSE: To investigate factors associated with self-care behaviors in individuals with diabetes in the community. METHODS: Researchers used secondary data analysis to assess survey data on diabetic individuals obtained from the Community Integrated Screening conducted by the Meishan Township Public Health Center and adult health examinations. Descriptive statistics and multiple linear regression analyses evaluated the independent factors associated with self-care behaviors. RESULTS: A total of ninety-seven individuals with Type 2 diabetes met the inclusion criteria. Mean age was 67.1 years and 37.1% was male. Results from multiple linear regression analysis indicated self-care behavior as poorer in individuals who drank alcohol during the past six months (p=.001), smoked cigarettes during the past six months (p=.015), and/or practiced religious or spiritual beliefs (p=.003). CONCLUSIONS/IMPLICATIONS FOR PRACTICE: Findings from this study can help understand factors affecting self-care behaviors in individuals with diabetes in the community. Medical and public health units should focus on providing effective advice on smoking and drinking cessation to individuals with diabetes. Units should also clarify potential conflicts between individuals' self-care behaviors and their religious or spiritual beliefs.

Doxycycline suppresses doxorubicin-induced oxidative stress and cellular apoptosis in mouse hearts.

Cardiac toxicity remains a serious yet unsolved complication of doxorubicin. This study was designed to examine whether doxycycline, a tetracycline-derived synthetic antibiotic with potential cytoprotective properties, could ameliorate this complication of doxorubicin. Male mice at 4-week of age were administrated with vehicle, doxorubicin (3mg/kg intraperitoneally every other day at 3 doses), doxycycline (2.5mg/kg intraperitoneally every other day for 3 doses), or doxycycline plus doxorubicin (each dose given 1day post doxycycline). After 28days, left ventricular geometric and systolic parameters were measured by transthoracic echocardiography, and hearts were harvested for extensive analyses regarding oxidative stress and cellular apoptosis. At 28days, hearts of doxorubicin-treated mice were characterized by less weight compared with controls, also with remodeling and depressed systolic function of the left ventricle. Biochemical analyses disclosed that content of malondialdehyde was increased and activity of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase, was decreased in these hearts. Both mitochondrion-dependent and endoplasmic reticulum stress-induced apoptotic pathways were also activated in the hearts of doxorubicin-treated mice as reflected by decreased Bcl-2/Bcl-(XL) and elevated Bax/Bad, p53/Apaf-1, endoplasmic reticulum glucose-related protein 78, C/EBP homologous protein, cytochrome c release from mitochondria, caspases-9/-3 cleavage, and cardiomyocyte apoptosis. In contrast, all the above left ventricular remodeling, systolic depressing, oxidative and pro-apoptotic actions of doxorubicin could be significantly alleviated by doxycycline pretreatment. Thus, doxycycline extensively counteracts multiple oxidative and apoptotic actions of doxorubicin in heart, hence may serve as an adjuvant agent to assuage the untoward cardiac effects of doxorubicin in clinical application.

The effects of overexpression of histamine releasing factor (HRF) in a transgenic mouse model.

BACKGROUND: Asthma is a disease that affects all ages, races and ethnic groups. Its incidence is increasing both in Westernized countries and underdeveloped countries. It involves inflammation, genetics and environment and therefore, proteins that exacerbate the asthmatic, allergic phenotype are important. Our laboratory purified and cloned a histamine releasing factor (HRF) that was a complete stimulus for histamine and IL-4 secretion from a subpopulation of allergic donors' basophils. Throughout the course of studying HRF, it was uncovered that HRF enhances or primes histamine release and IL-13 production from all anti-IgE antibody stimulated basophils. In order to further delineate the biology of HRF, we generated a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: We constructed an inducible transgenic mouse model with HRF targeted to lung epithelial cells, via the Clara cells. In antigen naïve mice, overproduction of HRF yielded increases in BAL macrophages and statistical increases in mRNA levels for MCP-1 in the HRF transgenic mice compared to littermate controls. In addition to demonstrating intracellular HRF in the lung epithelial cells, we have also been able to document HRF's presence extracellularly in the BAL fluid of these transgenic mice. Furthermore, in the OVA challenged model, we show that HRF exacerbates the allergic, asthmatic responses. We found statistically significant increases in serum and BAL IgE, IL-4 protein and eosinophils in transgenic mice compared to controls. CONCLUSIONS/SIGNIFICANCE: This mouse model demonstrates that HRF expression enhances allergic, asthmatic inflammation and can now be used as a tool to further dissect the biology of HRF.

EGCG protects against oxidized LDL-induced endothelial dysfunction by inhibiting LOX-1-mediated signaling.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), originally identified as the major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells, plays a major role in the pathology of vascular diseases. Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. In the present study, we hypothesized that the most abundant polyphenolic compound in tea, epigallocatechin-3-gallate (EGCG), can downregulate parameters of endothelial dysfunction by modulating LOX-1-regulated cell signaling. In cultured human umbilical vein endothelial cells (HUVECs), exposure to oxLDL (130 microg/ml), which led to an increase in LOX-1 expression at the RNA and protein levels, was abrogated by addition of EGCG or DPI, a well-known inhibitor of flavoproteins, suggesting the involvement of NADPH oxidase. Furthermore, oxLDL rapidly activated the membrane translocation of Rac-1 and p47phox and the subsequent induction of ROS generation, which was suppressed markedly by pretreatment with EGCG or anti-LOX-1 monoclonal antibody. OxLDL also increased p38 MAPK phosphorylation and decreased phosphorylation of the amino-terminal region of Akt, with maximal induction at about 30 min, and NF-kappaB phosphorylation within 1 h, resulting in redox-sensitive signaling. In addition, oxLDL diminished the expression of endothelial nitric oxide synthase (eNOS), enhanced the expression of endothelin-1 and adhesion molecules (ICAM, E-selectin, and monocyte chemoattractant protein-1), and increased the adherence of monocytic THP-1 cells to HUVECs. Pretreatment with EGCG, however, exerted significant cytoprotective effects in all events. These data suggest that EGCG inhibits the oxLDL-induced LOX-1-mediated signaling pathway, at least in part, by inhibiting NADPH oxidase and consequent ROS-enhanced LOX-1 expression, which contributes to further ROS generation and the subsequent activation of NF-kappaB via the p38 MAPK pathway. Results from this study may provide insight into a possible molecular mechanism by which EGCG suppresses oxLDL-mediated vascular endothelial dysfunction.

Ginkgo biloba extract 761 reduces doxorubicin-induced apoptotic damage in rat hearts and neonatal cardiomyocytes.

AIMS: The objective of this study was to investigate whether a cytoprotective herb-derived agent, Ginkgo biloba extract (EGb) 761, could have a beneficial effect on doxorubicin-induced cardiac toxicity in vitro and in vivo. METHODS AND RESULTS: Primary cultured neonatal rat cardiomyocytes were treated with the vehicle, doxorubicin (1 microM), EGb761 (25 microg/mL), or EGb761 plus doxorubicin. After 24 h, doxorubicin upregulated p53 mRNA expression, disturbed Bcl-2 family protein balance, disrupted mitochondrial membrane potential, precipitated mitochondrion-dependent apoptotic signalling, induced apoptotic cell death, and reduced viability of cardiomyocytes, whereas EGb761 pretreatment suppressed all the actions of doxorubicin. Similarly, rats treated with doxorubicin [3 mg/kg intraperitoneally (i.p.) three doses every other day] displayed retarded growth of body and heart as well as elevated apoptotic indexes in heart tissue at both 7 and 28 days after exposure, whereas EGb761 pretreatment (5 mg/kg i.p. 1 day before each dose of doxorubicin) effectively neutralized the aforementioned gross and cellular adverse effects of doxorubicin. CONCLUSION: Doxorubicin impairs viability of cardiomyocytes at least partially by activating the p53-mediated, mitochondrion-dependent apoptotic signalling. EGb761 can effectively and extensively counteract this action of doxorubicin, and may potentially protect the heart from the severe toxicity of doxorubicin.

Protection by doxycycline against doxorubicin-induced oxidative stress and apoptosis in mouse testes.

Spermatogenic cells constitute one of the body tissues that are susceptible to doxorubicin-induced oxidative stress and apoptosis. To explore whether doxorubicin toxicity to these male germ cells could be prevented by adjuvant medication, this study was designed to examine the possible ameliorating action of doxycycline, an antibiotic with anti-oxidant property, on doxorubicin-induced oxidative and apoptotic effects in mouse testes. Male mice at 5-week of age were treated with vehicles, doxorubicin alone (3 mg/kg, i.p. every other day for 3 doses), doxycycline alone (2.5 mg/kg, i.p. every other day for 3 doses), or doxycycline plus doxorubicin (each dose given 1 day post-doxycycline). After 28 days, mice treated with doxorubicin alone displayed smaller body and testicular weights, reduced sperm counts, impaired spermatogenic capability (scarcer spermatids and spermatocytes), increased oxidative stress (malondialdehyde levels), decreased anti-oxidant activity (superoxide dismutase and glutathione peroxidase), and elevated apoptotic indexes (upregulation of Bax and Bad, downregulation of Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to cytosol, activation of caspase-3, and increase of cleaved caspase-3 abundance and TUNEL positive cells), while doxycycline pretreatment could effectively prevent nearly all of these abnormalities. These results provide firm evidence that doxycycline pretreatment would offset the oxidative and apoptotic impact imposed by doxorubicin, and imply doxycycline to be a promising adjuvant agent that may attenuate the toxicity of doxorubicin on testicular tissues in clinical practice.

Stage-dependent expression of extra-embryonic tissue-spermatogenesis-homeobox gene 1 (ESX1) protein, a candidate marker for X chromosome-bearing sperm.

Extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) encodes an X-linked homeobox protein. Despite the fact that the temporal and spatial mRNA expression pattern of the protein has been studied extensively in the testis, specific localisation of ESX1 in the testis remains to be determined. In the present study, we generated ESX1 antiserum to investigate the stage- and tissue-specific expression of ESX1 in the mouse. Western blotting and immunofluorescent analyses revealed that general localisations of ESX1 were consistent with its RNA expression patterns; that is, it was restricted mainly to the placenta and testis. Immunofluorescent studies demonstrated that ESX1 existed in the testes after 3 weeks of age, coincident with the appearance of round spermatids in the seminiferous tubules. Moreover, ESX1 expression became more abundant in the luminal regions of the seminiferous tubules as the development of round spermatids progressed into spermatozoa. In contrast, reduced expression of ESX1 was observed in experimentally induced cryptorchid testes. The later expression of ESX1 suggests a role in post-meiotic germ cell development. To further understand ESX1 expression in sperm with respect to X chromosome-bearing sperm, we used ESX1 antiserum to immunostain sperm by confocal laser microscopy. Approximately half the sperm population was recognised by the ESX1 antiserum. On the basis of results of the present study, we suggest that ESX1 could be used as a protein marker for X chromosome-bearing sperm.