Dietary supplementation of soy germ phytoestrogens or estradiol improves spatial memory performance and increases gene expression of BDNF, TrkB receptor and synaptic factors in ovariectomized rats.

BACKGROUND: Estrogen or phytoestrogens treatment has been suggested to improve cognitive function of the brain in postmenopausal women. However, there is lack of information on the mechanism of such treatment on the central nervous system. The present study aimed to determine the effects of estradiol and soy germ phytoestrogens on spatial memory performance in ovariectomized rats and to explore the underlying mechanisms affecting the central nervous system. METHODS: Ovariectomized Sprague-Dawley rats were fed a basic diet supplemented with soy germ phytoestrogens (0.4 g/kg or 1.6 g/kg) or 17ß-estradiol (0.15 g/kg) for 12 weeks. At the end of the experiment, animals were evaluated for their spatial learning and memory performance by the Morris Water Maze task. The expressions of brain-derived neurotrophic factor (BDNF) and synaptic formation proteins in the hippocampal tissue were estimated using RT-PCR and ELISA. RESULTS: It was found that rats supplemented with soy germ phytoestrogens or estradiol performed significantly better in spatial memory acquisition and retention when compared to the rats fed on the control diet. Estradiol or the high dose of phytoestrogens treatment significantly increased BDNF concentration and the mRNA levels for BDNF and its TrkB receptors as well as the synaptic formation proteins, synaptophysin, spinophilin, synapsin 1 and PSD-95, in the hippocampal tissue of the experimental animals. It was also found that phytoestrogens, in contrast to estradiol, did not show any significant effect on the vaginal and uteri. CONCLUSION: Soy germ phytoestrogens, which may be a substitute of estradiol, improved spatial memory performance in ovariectomized rats without significant side-effects on the vaginal and uteri. The memory enhancement effect may relate to the increase in BDNF and the synaptic formation proteins expression in the hippocampus of the brain.

Lycium barbarum polysaccharides induce apoptosis in human prostate cancer cells and inhibits prostate cancer growth in a xenograft mouse model of human prostate cancer.

Lycium barbarum polysaccharides (LBPs) are important functional constituents in red-colored fruits of L. barbarum (Guo Qi Zi, a well-known traditional Chinese medicinal plant commonly known as Goji berry or wolfberry). The influence of LBP on human prostate cancer cells was systematically investigated in vitro and in vivo. The in vitro effects of LBP on two cell lines (PC-3 and DU-145) were examined by using trypan blue exclusion staining, single-cell gel electrophoresis, flow cytometry, terminal dUTP nick-end labeling assay, and immunohistochemical assay (assessment of Bcl-2 and Bax expression). The in vivo effect of LBP on PC-3 cells was assessed in the nude mouse xenograft tumor model. The in vitro results showed that LBP can dose- and time-dependently inhibit the growth of both PC-3 and DU-145 cells. LBP caused the breakage of DNA strands of PC-3 and DU-145 cells; the tail frequency and tail length were significantly higher than that of control cells. LBP also markedly induced PC-3 and DU-145 cell apoptosis, with the highest apoptosis rates at 41.5% and 35.5%, respectively. The ratio of Bcl-2/Bax protein expression following LBP treatments decreased significantly with a dose-effect relationship, which suggested that LBP can regulate the expression of Bcl-2 and Bax to induce apoptosis of PC-3 and DU-145 cells. The in vivo experimental results indicate that LBP might significantly inhibit PC-3 tumor growth in nude mice. Both the tumor volume and weight of the LBP treatment group were significantly lower than those of the control group.

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells.

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2'-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation.

Immunolocalization assessment of metastasis-associated protein 1 in human and mouse mature testes and its association with spermatogenesis.

AIM: To investigate the stage-specific localization of metastasis-associated protein 1 (MTA1) during spermatogenesis in adult human and mouse testis. METHODS: The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. RESULTS: The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species: in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. CONCLUSION: The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.

The effects of perinatal protein malnutrition on spatial learning and memory behaviour and brain-derived neurotrophic factor concentration in the brain tissue in young rats.

This study aimed to investigate the effects of perinatal protein malnutrition on brain derived-neurotrophic factor (BDNF) concentration in brain tissue and spatial learning and memory performance in young rats. Nine pregnant Wistar rats were assigned into three groups. Rats in one group were fed with a control diet containing 20% protein. Rats in remaining two groups were fed with a diet containing 6% protein from gestation day eight and day 15 respectively till four weeks after birth. At four weeks of age, the rat pups were evaluated for spatial learning ability using Morris Water Maze (MWM) task. At the end of the behaviour tests, rat pups were sacrificed and the brain tissue samples were collected for measurement of total protein and BDNF concentrations. It was found that rat pups fed the low protein diet had lower body weight and slightly lighter brain compared to the control pups. Total protein levels in hippocampus and cerebral cortex were significantly lower in malnourished pups than the controls. The concentration of BDNF in the hippocampus was also significantly lower in rat pups suffered protein malnutrition from early pregnancy than in the controls. MWM tests showed that perinatal protein deprivation, particularly from early pregnancy, significantly impaired learning and memory ability. The results of the present study indicate that perinatal protein malnutrition had adverse influence on spatial navigation and brain BDNF levels in rats. The decreased hippocampal BDNF concentration might partially contribute to the poor learning memory performance in the protein deprived rats.

Serial changes in urinary proteome profile of membranous nephropathy: implications for pathophysiology and biomarker discovery.

Membranous nephropathy is one of the most common causes of primary glomerular diseases worldwide. The present study adopted a gel-based proteomics approach to better understand the pathophysiology and define biomarker candidates of human membranous nephropathy using an animal model of passive Heymann nephritis (PHN). Clinical characteristics of Sprague-Dawley rats injected with rabbit anti-Fx1A antiserum mimicked those of human membranous nephropathy. Serial urine samples were collected at Days 0, 10, 20, 30, 40, and 50 after the injection with anti-Fx1A (number of rats = 6; total number of gels = 36). Urinary proteome profiles were examined using 2D-PAGE and SYPRO Ruby staining. Quantitative intensity analysis and ANOVA with Tukey post-hoc multiple comparisons revealed 37 differentially expressed proteins among 6 different time-points. These altered proteins were successfully identified by MALDI-TOF MS and classified into 6 categories: (i) proteins with decreased urinary excretion during PHN; (ii) proteins with increased urinary excretion during PHN; (iii) proteins with increased urinary excretion during PHN, but which finally returned to basal levels; (iv) proteins with increased urinary excretion during PHN, but which finally declined below basal levels; (v) proteins with undetectable levels in the urine during PHN; and (vi) proteins that were detectable in the urine only during PHN. Most of these altered proteins have functional significance in signaling pathways, glomerular trafficking, and controlling the glomerular permeability. The ones in categories (v) and (vi) may serve as biomarkers for detecting or monitoring membranous nephropathy. After normalization of the data with 24-h urine creatinine excretion, changes in 34 of initially 37 differentially expressed proteins remained statistically significant. These data underscore the significant impact of urinary proteomics in unraveling disease pathophysiology and biomarker discovery.

Dietary insulin affects leucine aminopeptidase, growth hormone, insulin-like growth factor I and insulin receptors in the intestinal mucosa of neonatal pigs.

BACKGROUND: Recent studies suggest that milk-borne insulin may regulate the development of the gastrointestinal tract in neonatal mammals. OBJECTIVES: To explore the mechanism by which milk-borne insulin affects gastrointestinal tract development, we examined the effect of dietary insulin on the expression levels of leucine aminopeptidase (LAP) and insulin-like growth factor I (IGF-I), as well as its effect on growth hormone (GH), IGF-I and insulin receptors in the small intestinal mucosa of neonatal pigs. METHODS: Five piglets were anesthetized and sampled within 2-4 h after birth. They were not allowed to suckle and were used as newborn controls (group N). Ten other piglets from 5 litters were randomly divided into group M (n=5), which was fed cow's milk, and group MI (n=5), which was fed cow's milk and insulin (2.5 mg/l). Piglets in groups M and MI were artificially fed for 3 days and then sampled. Total RNA in their intestinal mucosa was extracted with Tripure reagents (Roche, USA). Reverse transcription PCR (RT-PCR) was used to semi-quantify mRNA levels of target genes and 18S rRNA was used in an RT-PCR system as an internal control. PCR products were loaded onto a 9% nondenaturing polyacrylamide gel. The gel was stained by silver staining agents. Digital photos were taken and the strength of the band areas was quantified using software. RESULTS: The results showed that the DNA contents and LAP activity in the small intestines of the piglets in group MI were higher (p<0.05) than in the piglets in group N. Compared with group M, piglets in group MI exhibited significantly increased expression levels of both insulin and GH receptor in the ileum, and LAP in the jejunum (p<0.05); IGF-I receptor expression levels in both the jejunum and ileum were significantly decreased (p<0.01 and p<0.05, respectively), while IGF-I expression was unchanged (p>0.05). CONCLUSION: Collectively, dietary insulin increased mRNA levels of insulin and GH receptor, which could help explain the effect of dietary insulin on receptor-mediated postnatal development of the small intestine. Dietary insulin suppressed IGF-I receptor expression, which may be the result of negative feedback caused when insulin binds to IGF-I receptors.

DHA depletion in rat brain is associated with impairment on spatial learning and memory.

OBJECTIVE: To examine the effect of docosahexaenoic acid (DHA) deficiency in brain on spatial learning and memory in rats. METHODS: Sprague Dawley rats were fed with an n-3 fatty acid deficient diet for two generations to induce DHA depletion in brain. DHA in seven brain regions was analyzed using the gas-liquid chromatography. Morris water maze (MWM) was employed as an assessing index of spatial learning and memory in the n-3 fatty acid deficient adult rats of second generation. RESULTS: Feeding an n-3 deficient diet for two generations depleted DHA differently by 39%-63% in the seven brain regions including cerebellum, medulla, hypothalamus, striatum, hippocampus, cortex and midbrain. The MWM test showed that the n-3 deficient rats took a longer time and swam a longer distance to find the escape platform than the n-3 Adq group. CONCLUSION: The spatial learning and memory in adult rats are partially impaired by brain DHA depletion.

Effects of intrauterine growth retardation on development of the gastrointestinal tract in neonatal pigs.

BACKGROUND: Intrauterine growth retardation (IUGR) is a common problem in human and other species and increases the risk of death of the fetus and newborn during the perinatal period. OBJECTIVES: This study was conducted to examine the influences of intrauterine growth retardation (IUGR) on development of the gastrointestinal tract in newborn pigs. METHODS: Ten animals from five litters were divided into five piglets with IUGR and five with normal birth-weight (NW). The IUGR category comprised animals with a birth weight 2 SD below the mean birth weight of the total population, while the NW category included animals with a birth weight within one SD of the mean birth weight in the total population. Animals were anesthetized and sampled within 2-4 h after birth and without suckling. The morphological changes of intestine and stomach of IUGR piglets were compared with NW ones. The expressions of IGF-I and receptors for growth hormone and insulin in intestinal mucosa were semiquantified using reverse transcription PCR. RESULTS: The results of our study indicated that the weights of the stomach, small intestine and small intestinal mucosa were significantly lower in IUGR compared with NW piglets (p<0.01). In addition, the lengths of the small intestine and colon in IUGR pigs were also significantly less than those of NW (p<0.05). Furthermore, insulin-like growth factor-I (IGF-I) mRNA level in intestinal mucosa of IUGR piglets was increased significantly (p<0.05), and the expression mRNA levels of insulin receptor and growth hormone (GH) receptor in the mucosa in IUGR piglets showed a tendency to be lower (p=0.17 and p=0.11, respectively) than those of the NW animals. CONCLUSION: We conclude from the data that IUGR affects intestinal growth and morphology and is in associated with altered gene expression of growth-related proteins. We speculate that the morphological change and associated altered endocrine homeostasis contribute to lower growth rates of pigs affected by IUGR.

Transient changes of transforming growth factor-beta expression in the small intestine of the pig in association with weaning.

It is well known that early weaning causes marked changes in intestinal structure and function, and transforming growth factor-beta (TGF-beta) is believed to play an important regulatory role in post-weaning adaptation of the small intestine. The present study examined the distribution and expression intensity of TGF-beta in the small intestinal mucosa of pre- and post-weaning pigs using a specific immunostaining technique and Western blot analysis. The level of TGF-beta in the intestinal mucosa, as estimated by Western blot analysis, did not change significantly during weaning. However, when examined by the immunostaining technique, TGF-beta1 (one of the TGF-beta isoforms dominantly expressed in the tissue) at the intestinal villus epithelium, particularly at the apical membrane of the epithelium, decreased significantly 4 d after weaning, while the staining intensity increased significantly at the intestinal crypts compared with that in pre-weaning pigs. These changes were transient, with the immunostaining intensity for TGF-beta1 at the intestinal villi and the crypts returning to the pre-weaning level by 8 d post-weaning. The transient decrease in TGF-beta1 level at the intestinal villus epithelium was associated with obvious intestinal villus atrophy and marked reduction of mucosal digestive enzyme activities. Furthermore, the number of leucocytes staining positively for TGF-beta1 increased significantly in the pig intestinal lamina propria 4 d after weaning. These findings strongly suggest that TGF-beta plays an important role in the post-weaning adaptation process in the intestine of the pig.

Stage-specific localization of transforming growth factor beta1 and beta3 and their receptors during spermatogenesis in men.

AIM: To investigate the stage-specific localization of transforming growth factor (TGF) beta1 and beta3 during spermatogenesis in adult human testis. METHODS: The localization of TGFbeta1 and beta3 was investigated by immunohistochemical staining method employing specific polyclonal antibodies. RESULTS: Both TGFbeta1 and beta3 and their receptors were preponderant in the Leydig cells. TGFbeta1 could not be detected in the seminiferous tubules. TGFbeta3 and TGFbeta-Receptor (R) I were mainly seen in the elongated spermatids, while TGFbeta-RII in the pachytene spermatocytes and weak in the spermatogonia, spermatids and Sertoli cells. Only TGFbeta-RII was detected in the Sertoli cells. TGFbeta3, TGFbeta-RI and TGFbeta-RII showed a staining pattern dependent upon the stages of the seminiferous epithelium cycle. CONCLUSION: TGFbeta isoforms and their receptors are present in the somatic and germ cells of the adult human testis, suggesting their involvement in the regulation of spermatogenesis.

Expression and localization of Smad1, Smad2 and Smad4 proteins in rat testis during postnatal development.

AIM: To study the expression and regulation of Smad1, Smad2 and Smad4 proteins (intracellular signaling molecules of transforming growth factor-b family) in rat testis during postnatal development. METHODS: The whole testes were collected from SD rats aged 3, 7, 14, 28 and 90 (adult) days. The cellular localization and developmental changes were examined by immunohistochemistry ABC method with the glucose oxidase-DAB-nickel enhancement technique. Quantitative analysis of the immunostaining was made by the image analysis system. The Smads proteins coexistence in the adult rat testis was tested by the double immune staining for CD14-Smad4 and Smad2-Smad4. The protein expression of Smad during rat testicular development was examined by means of Western blots. RESULTS: Smad1, Smad2 and Smad4 were present throughout testicular development. The immunostaining of Smad1 and Smad2 were present in spermatogenic cells. A positive immunoreactivity was located at the cytoplasm, but the nucleus was negative. Smad1 was immunolocalized at the d14, d28 and adult testes, while Smad2, at the d7, d14, d28 and adult testis. There was positive immunoreaction in the Sertoli cells and Leydig cells as well. The immunolocalization of Smad4 was exclusively at the cytoplasm of Leydig cells and the nuclei were negative throughout the testicular development. No expression was detected in the germ cells. The results of image and statistical analysis showed that generally the expression of Smad1, Smad2 and Smad4 in the testis tended to increase gradually with the growth of the rat. CONCLUSION: The present data provide direct evidences for the molecular mechanism of TGF-bgr action in rat testes during postnatal development and spermatogenesis.

[Location studies of Smad4 protein in the rat testis during postnatal development].

In order to shed light on the mechanisms of TGF-beta action in the testis,we examined the expression and function of Smad4 protein, the common-mediator Smads, which is one of intracellular signaling molecules of TGF-beta superfamily members, in rat testis during postnatal development. Whole testes were collected from SD rats aged 3 days, 7 days, 14 days and 28 days, and adult. In this study, we examined, by means of western blots, the protein expression of Smad4 during rat testicular development and its cellular localization by immunohistochemical ABC method with glucose oxidase-DAB-nickel enhancement technique. The results showed that the protein of Smad4 was present in rats from 3 days of age to adulthood, and the immunoreactivity for Smad4 was exclusively localized to the cytoplasm of Leydig cells with negative nuclei in the interstitial tissue at any time point. No expression was detected in germ cells. Therefore, our data provide evidence for the molecular mechanism of TGF-beta action in rat testes during postnatal development and spermatogenesis of rats.