Cryptosporidium parvum at different developmental stages modulates host cell apoptosis in vitro

We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.     

Fas expression correlates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis

Degeneration of human male germ cells was analysed by means of light (LM) and transmission electron (TEM) microscopy. The frequency of degenerating cells was correlated with that of Fas-expressing germ cells in human testes with normal spermatogenesis (n = 10), complete early maturation arrest (EMA) (n = 10) or incomplete late maturation arrest (LMA; n = 10) of spermatogenesis. LM analysis of testis sections with normal spermatogenesis indicated that degenerating germ cells were localized in the adluminal compartment of the seminiferous epithelium. TEM showed that apoptotic cells were mostly primary spermatocytes and, to a lesser extent, round or early elongating spermatids. Apoptotic germ cells appeared to be eliminated either in the seminiferous lumen or by Sertoli cell phagocytosis. An increased number of degenerating cells was observed in testes with LMA as compared with normal testes and testes with EMA of spermatogenesis (P < 0.001, Wilcoxon's rank sum test). Comparison of these results with those obtained from immunohistochemistry experiments demonstrated a tight correlation between the number of apoptotic cells and the number of Fas-expressing germ cells (P = 0.001, Spearman's rank = 0.69). These findings suggest that altered meiotic and post-meiotic germ cell maturation might be associated with an up-regulation of Fas gene expression capable of triggering apoptotic elimination of defective germ cells.     

Low dose bisphenol A impairs spermatogenesis by suppressing reproductive hormone production and promoting germ cell apoptosis in adult rats

Bisphenol A (BPA), an estrogenic chemical, has been shown to reduce sperm count; however, the underlying mechanisms remain unknown. Herein, we show that oral administration of BPA (2 µg/kg) for consecutive 14 days in adult rats (BPA rats) significantly reduced the sperm count and the number of germ cells compared to controls. The serum levels of testosterone and follicle-stimulating hormone (FSH), as well as the level of GnRH mRNA in BPA rats were lower than those of control rats. Testosterone treatment could partially rescue the reduction of germ cells in BPA rats. Notably, the number of apoptotic germ cells was significantly increased in BPA rats, which was insensitive to testosterone. Furthermore, the levels of Fas, FasL and caspase-3 mRNA in the testicle of BPA rats were increased in comparison with controls. These results indicate that exposure to a low dose of BPA impairs spermatogenesis through decreasing reproductive hormones and activating the Fas/FasL signaling pathway.     

Somatic cell apoptosis markers and pathways in human ejaculated sperm: potential utility as indicators of sperm quality

In this study we extended earlier work to determine whether sperm respond to somatic cell apoptotic stimuli and whether apoptotic phenotypes are significant indicators of human sperm quality. We evaluated ejaculated sperm from fertile donors and subfertile patients following purification of fractions of high and low motility. In unstimulated conditions, caspase enzymatic activity was higher in motile fractions from subfertile patients than in donors, and was higher in low motility fractions from both groups. Staurosporine, but not a Fas ligand or H2O2, significantly increased caspase activity, but only in high motility fractions. Procaspase-3, -7 and -9 and low levels of active caspase-3, -7 and -9 were identified by immunoblot analysis. Apoptosis-inducing factor (AIF) was present in all samples but poly ADP-ribose polymerase-1 (PARP-1) was not detected. Phosphatidylserine translocation was significantly increased only with H2O2 treatment. In ejaculates of both subfertile and fertile men, we demonstrated the presence and activation of several proteins that are key constituents of apoptosis-related pathways in somatic cells, which may serve as markers for sperm quality.     

Fine needle aspiration versus open biopsy for testicular sperm recovery: a controlled study in azoospermic patients with normal spermatogenesis

This retrospective controlled study aimed at comparing two techniques for recovering testicular spermatozoa in azoospermic patients undergoing intracytoplasmic sperm injection (ICSI). 102 men suffering from infertility because of obstructive azoospermia had ICSI using testicular spermatozoa recovered either by open excisional biopsy (n = 51), or by fine needle aspiration (FNA) (n = 51). A higher average number of spermatozoa were recovered after open biopsy than after FNA, but no significant differences in either fertilization rates or cleavage rates were observed after ICSI with spermatozoa retrieved by the two techniques. Neither was there any significant difference in ongoing pregnancy and implantation rates: in the FNA group, these figures were respectively 19.6% per cycle and 7.8% per embryo transferred and in the open biopsy group 21.6 and 7.1%. We conclude that ICSI with testicular spermatozoa recovered by FNA yields results comparable to those obtained with spermatozoa recovered by open biopsy in azoospermic patients with normal spermatogenesis. However a prospective study is needed to confirm the present results and to assess recovery rates and patient comfort for the two methods.      

Openings in frog microvascular endothelium at different rates of increase in pressure and at different temperatures

Experiments were carried out on single mesenteric capillaries and venules of pithed frogs to determine whether the rate of increase in intravascular pressure (d P /d t ) influenced the critical pressure ( P _B) which increases wall permeability. Vessels, microperfused with frog Ringer solutions containing 0.1 % bovine serum albumin and red cells, were occluded downstream before pressure was raised either as a ramp or in a series of 13.6 cmH₂O steps. By varying step duration, the mean d P /d t could be matched to d P /d t applied as a steady ramp. P _B was recorded as the pressure at which there was an abrupt increase in filtration with red cells passing to and through one or more sites in the vessel wall. In all vessels, increasing d P /d t raised P _B, with no differences between steps and ramps. The relation between P _B and d P /d t was linear, consistent with a latent period, T (the slope), between a critical pressure being reached and the abrupt increase in permeability being observed. Direct observation confirmed this latent period. Between 12 and 20 ^oC, T was 8.5 ± 0.47 s; between 0 and 5 °C, T was 11.5 ± 0.97 s. Tissue cooling did not influence the time constant, τ, describing the rate of stretch of wall following a step increase in pressure and used to measure wall visco-elastic properties. Nor was the value of τ (1.15 ± 0.06 s, n = 42) consistent with T being accounted for by visco-elasticity. It is suggested that the latent period may indicate an active response of the endothelium.     

Influence of aging on mast cell production of a nitric oxide-like factor: Comparison between normal and hypertensive rats

Rat serosal mast cells (MCs) isolated from Wistar albino rats synthesize a nitric oxide (NO)-like factor which inhibits platelet aggregation, stimulates guanylate cyclase and modulates mast cell histamine release. Aging can affect the ability of MCs to generate and release chemical mediators.The aim of the present study was to evaluate the ability to generate a NO-like factor by MCs isolated from 2, 6 and 18 month old normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats (systolic blood pressure 180±2.5 mm Hg) by two bioassays: inhibition of platelet aggregation and stimulation of MC guanylate cyclase.     

Influence of aging on mast cell production of a nitric oxide-like factor: Comparison between normal and hypertensive rats

Rat serosal mast cells (MCs) isolated from Wistar albino rats synthesize a nitric oxide (NO)-like factor which inhibits platelet aggregation, stimulates guanylate cyclase and modulates mast cell histamine release. Aging can affect the ability of MCs to generate and release chemical mediators.

The aim of the present study was to evaluate the ability to generate a NO-like factor by MCs isolated from 2, 6 and 18 month old normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats (systolic blood pressure 180±2.5 mm Hg) by two bioassays: inhibition of platelet aggregation and stimulation of MC guanylate cyclase.

MCs from SH rats produced less NO-like activity than MCs from normotensive WKY rats of the same age. In both strains aging decreased the capacity of MCs to generate the NO-like factor. These results suggest that NO synthesis and/or release from MCs is involved in some events associated with aging and hypertension.

     

Surface interaction in vitro between sertoli cells and germ cells at different stages of spermatogenesis

The presence of surface-recognition mechanisms between somatic and germ cells of the seminiferous epithelium has been studied in the rat by an assay in vitro based on the ability of homogeneous populations of spermatogenic cells, at specific differentiative stages, to adhere to monolayers of cultured Sertoli cells. The results show that germ cells adhere specifically to Sertoli cells and that the adhesion is dependent on the differentiative stage of the germ cells. Pachytene spermatocytes show the highest ability to adhere and form typical junctional specializations with the underlying Sertoli cells, while round spermatids adhere much less to the substrate. The possible regulative role of a somatic cell-germ cell interaction is discussed.     

Germ cell loss in Klinefelter syndrome: When and why?

Klinefelter syndrome (KS) is a quite common disorder with an incidence of 1–2 in 1,000 new‐born males. Most patients are diagnosed in the light of a clinical checkup when consulting a fertility clinic with an unfulfilled child wish. Infertility in KS patients is caused by a massive germ cell loss, leading to azoospermia in more than 90% of the adult patients. Most seminiferous tubules in the adult KS testis are degenerated or hyalinized and testicular fibrosis can be observed, starting from puberty. However, focal spermatogenesis can be found in the testis of some patients. This offers the opportunity to extract spermatozoa from the testis by testicular sperm extraction (TESE). Nevertheless, TESE is only successful in about half of the KS adults seeking to father children. The reason for the germ cell loss remains unclear. To date, it is still debated whether the testicular tissue changes and the germ cell loss seen in KS is directly caused by an altered X‐linked gene expression, the altered somatic environment, or a deficiency in the germ cells. In this review, we provide an overview of the current knowledge about the germ cell loss in KS patients.     

Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from

T cells developing in the thymus are derived from hematopoietic stem cells (HSCs) in the bone marrow (BM). Understanding the developmental steps linking multipotent HSCs to intrathymic T lineage-committed progenitors is important for understanding cancer in T lineage cells, improving T cell reconstitution after BM transplantation, and designing gene-therapy approaches to treat defective T cell development or function. Such an understanding may also help ameliorate immunological defects in aging. This review covers the differentiation steps between HSCs and committed T cell progenitors within the thymus.     

Tumour necrosis factor-alpha and interferon-gamma production measured at the single cell level in normal and inflamed human intestine.

The spot-ELISA technique has been used to enumerate the frequency of cells secreting tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), isolated from biopsies of normal intestine and from biopsies of children with inflammatory bowel disease. TNF-alpha production was undetectable in six out of 12 biopsies from normal intestine and in the other six biopsies it ranged from 60 to 580 TNF-alpha-secreting cells/10(6) isolated intestinal cells. In contrast, cells isolated from biopsies of children with Crohn's disease (n = 9) all showed elevated frequencies of TNF-alpha-secreting cells (500-12,000 secreting cells/10(6) cells). In ulcerative colitis, four out of eight children had increased production of TNF-alpha and in children with indeterminate colitis two out of three had elevated levels. There was no correlation between plasma TNF-alpha levels and the number of intestinal cells secreting TNF-alpha. In controls and all groups of patients IFN-gamma-secreting cells were uncommon. These results suggest that TNF-alpha is an important mediator of inflammation in the human gut, and, furthermore, may play a role in the growth failure frequently seen in children with inflammatory bowel disease.     

生后不同发育阶段大鼠卵巢内eNOS和iNOS的表达

目的　观察eNOS和iNOS在卵巢中的表达,并探讨其意义。 方法　采用免疫组化和图像分析系统检测大鼠生后各发育阶段卵巢中eNOS和iNOS的定位分布和表达。 结果　0 d大鼠卵巢卵母细胞eNOS和iNOS均呈弱阳性反应;4 d后,eNOS和iNOS在正常卵母细胞中均呈强阳性,退化卵母细胞中反应非常弱;卵泡细胞与膜细胞中的iNOS在30 d前均呈强阳性表达,30 d后,生长卵泡中表达降低,闭锁卵泡中较正常生长卵泡表达增强,eNOS的表达稳定;240 d 后卵巢内间质成分和闭锁卵泡增多,间质中的iNOS表达增强。360d 后大鼠卵巢逐渐纤维化,iNOS染色强于eNOS。 结论　大鼠不同发育时期卵巢中,eNOS的表达相对稳定;iNOS的表达以性成熟后育龄期最低,随着年龄增加及卵巢功能的衰退,其表达逐渐增高。     

The production of IgE and IgGa antibodies in normal rats and rats infected with Nippostrongylus brasiliensis.

The time courses of production of IgE and IgGa homocytotropic antibodies were measured in Wistar rats during a primary and secondary response to egg albumin with pertussis or Freund's adjuvants. An anamnestic IgE antibody response occurred in animals previously sensitized to antigen with killed Bordetella pertussis as adjuvant. IgGa antibodies were formed in the primary response with Freund's complete adjuvant only, but were found during the secondary response with all adjuvants used. The time courses of formation of IgE and IgGa antibodies were very different during the secondary response. The production of both classes of antibody to egg albumin was studed in Wistar and Hooded Lister rats infected with Nippostrongylus brasiliensis. IgGa antibody formation was not potentiated by the infection. However, increased levels of IgE antibody, formed during a secondary response to antigen in infected animals, were consistently higher in both strains than during a primary response.     

A developmental analysis of clonidine's effects on cardiac rate and ultrasound production in infant rats

Under controlled conditions, infant rats emit ultrasonic vocalizations during extreme cold exposure and after administration of the alpha(2) adrenoceptor agonist, clonidine. Previous investigations have determined that, in response to clonidine, ultrasound production increases through the 2nd-week postpartum and decreases thereafter. Given that sympathetic neural dominance exhibits a similar developmental pattern, and given that clonidine induces sympathetic withdrawal and bradycardia, we hypothesized that clonidine's developmental effects on cardiac rate and ultrasound production would mirror each other. Therefore, in the present experiment, the effects of clonidine administration (0.5 mg/kg) on cardiac rate and ultrasound production were examined in 2-, 8-, 15-, and 20-day-old rats. Age-related changes in ultrasound production corresponded with changes in cardiovascular variables, including baseline cardiac rate and clonidine-induced bradycardia. This experiment is discussed with regard to the hypothesis that ultrasound production is the acoustic by-product of a physiological maneuver that compensates for clonidine's detrimental effects on cardiovascular function.