Immunization with ovarian autoantigens leads to reduced fertility in mice following follicular dysfunction

Immunoproteomics using sera of women with ovarian autoimmune diseases such as primary ovarian insufficiency and IVF embryo transfer recruits led to identification of three proteins namely alpha actinin 4 (α-ACTN4), heat-shock 70 protein 5 (HSPA5), and actin beta (ACTB). This study deals with the establishment of a peptide ELISA for screening sera of antiovarian antibody (AOA)-positive patients and further delves into understanding the role of these three proteins in ovarian autoimmunity in a mouse model. Using in silico approach, antigenic peptides of these proteins were identified and used for peptide ELISA. ELISA results indicated that AOA-positive sera showed reactivity with only specific peptides. The functional significance of the dominant peptides was studied by active immunization of female mice with these peptides. All immunized mice generated high antibody titers and profound effect on ovaries with few primordial (2.4±0.1, 2.4±0.2, and 2±0.1), primary (2.4±0.5, 1.7±0.3, and 2.4±0.3), preantral (2.3±0.5, 3.4±0.3, and 2.9±0.3), antral (0.9±0.2, 1.6±0.8, and 2.3±0.6) follicles, and corpora lutea (2.8±0.8, 2.9±1.7, and 4.6±2.3), and increased number of atretic follicles (5.5±0.4, 4.9±1.8, and 7.5±1.0) in ACTN4-, HSPA5-, and ACTB-immunized mice compared with control animals (3.0±0.2, 3.5±0.6, 3±0.1, 3.6±0.2, 4.7±0.3, and 1.5±0.3) respectively. These mice when mated with fertile male mice showed an overall 25-43% reduction in fertility compared with controls. The data clearly suggest that the dominant antigenic epitopes of the three proteins play critical role in fertility and could possibly be the key autoimmune targets. These epitopes could be used to develop a more specific and sensitive diagnostic test for women with ovarian autoimmune diseases and to design therapy for disease management for reinstatement of ovarian function.     

Age-related uterine and ovarian hypertrophy in FSH receptor knockout and FSHbeta subunit knockout mice

Female mice in which the gene encoding the follicle-stimulating hormone FSH receptor (FSHR) knockout (KO) or its ligand (FSHbetaKO) have been disrupted were infertile. Ovaries of these mice were significantly smaller than those of heterozygous littermates but significantly larger than those of hypogonadal mice of the same age. Uterine masses in all three mutants were <6 mg, significantly reduced compared with heterozygous mice. At 1 year of age uterine mass had increased to >12 mg in 63% of FSHRKO females and 88% of FSHbetaKO females. Despite the increase in uterine size there was no evidence of contractility: uteri were flaccid and unresponsive to electrical or pharmacological stimulation. In most females in which uterine growth had occurred there was evidence of ovarian growth with hypertrophy of the interstitial tissue, occurrence of ovarian cysts and epithelial and tubular inclusions. There was no evidence of uterine or ovarian hypertrophy in hypogonadal (hpg) mice at any age or in 1 year old females in which the FSH mutations were bred onto the hpg background. There was an inverse correlation of plasma LH concentrations and uterine mass in 1 year old mutant females with uterine hypertrophy. Ovariectomy of both FSHRKO and FSHbetaKO females with large uteri resulted in decreased uterine mass and increased plasma concentration of LH. The number of mice with ovarian pathology, reminiscent of the serous ovarian adenocarcinomas found in humans, was significantly greater in the FSHbetaKO mice, indicating that the presence of an intact FSH receptor on ovarian cells of FSHbetaKO females may allow constitutive basal stimulation of the ovary, which is absent in mice lacking FSH receptors.     

Increasing amounts of crushed wheat fed with pasture hay reduced dietary fiber digestibility in lactating dairy cows

Sixteen cows in mid-lactation (milk yield of 23.8 ± 2.3 kg/d) were individually fed diets consisting of chopped perennial ryegrass hay, offered at 3 kg of dry matter (DM)/100 kg of body weight (BW), fed either alone or supplemented with amounts of crushed wheat ranging from 0.4 to 1.6 kg of DM/100 kg of BW (increasing at nominal intervals of 0.4 kg of DM/100 kg of BW; 5 nominal treatments in total). Three cows were allocated to each treatment except the mid-range wheat treatment, which had 4 cows. Results were analyzed by regression because the intake of the wheat by cows within treatments varied. The hay was used to reflect the characteristics of summer pastures in southeastern Australia. Feed intake and fecal output were measured to determine digestion coefficients, feeds were incubated in nylon bags in the rumen, and rumen variables were monitored. Estimates of metabolizable energy (ME) of the hay from in vivo or in vitro digestibility were also compared. The digestibility of neutral detergent fiber (NDF) was depressed linearly as the amount of crushed wheat consumed increased to 36% of DM intake. The extent to which negative associative effects on NDF digestion were associated with the hay could not be determined, as it was not possible to distinguish between the NDF from hay and that from wheat. However, acid detergent fiber (ADF) digestion also declined, suggesting that most of the response lay with the hay because ADF was negligible in the wheat. Most data indicated that effects of proportion of wheat in the diet on the utilization of consumed nutrients were small. Despite substitution of wheat for hay reducing the forage intake of cows, there was a positive linear effect on marginal milk responses (1.3 kg of energy-corrected milk/kg of DM wheat). Mean rumen fluid pH declined as the proportion of wheat in the diet increased. The lowest pH for any individual cow during a 24-h period was 5.4, and the amount of time that rumen fluid pH was <6.0 ranged from 0 to 14 h depending on the amount of wheat consumed. It was concluded that these perturbations of the rumen environment were probably sufficient to result in negative associative effects. In addition, estimates of the ME content of the hay were higher when calculated from in vitro compared with in vivo digestibility, which has implications when estimating the amount of feed required for production.     

Endocrine-disrupting chemicals in ovarian function: effects on steroidogenesis, metabolism and nuclear receptor signaling

Endocrine-disrupting chemicals (EDCs) are exogenous agents with the ability to interfere with processes regulated by endogenous hormones. One such process is female reproductive function. The major reproductive organ in the female is the ovary. Disruptions in ovarian processes by EDCs can lead to adverse outcomes such as anovulation, infertility, estrogen deficiency, and premature ovarian failure among others. This review summarizes the effects of EDCs on ovarian function by describing how they interfere with hormone signaling via two mechanisms: altering the availability of ovarian hormones, and altering binding and activity of the hormone at the receptor level. Among the chemicals covered are pesticides (e.g. dichlorodiphenyltrichloroethane and methoxychlor), plasticizers (e.g. bisphenol A and phthalates), dioxins, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons (e.g. benzo[a]pyrene).     

Dexamethasone influences endocrine and ovarian function in dairy cattle

Multiparous nonlactating Holstein cows were used to determine the effect of dexamethasone on ovarian follicular development and plasma hormone concentrations. Animals were randomly divided into two groups, control (C; n = 5) and treatment (T; n = 6), but managed as one group. Both groups were synchronized with two injections of PGF2alpha (25 mg i.m.) 11 d apart. One day after ovulation (d 0) the T group received a daily injection of dexamethasone (44 microg/kg of body weight; i.m.) until the first dominant follicle stopped growing or up to d 12 postovulation. The C group received vehicle injections. Blood samples were collected daily from all cows. Concentrations of LH and FSH did not differ between the C and T cows, whereas progesterone concentrations were lower in T than in C cows from d 4 onward. Treatment x day interaction influenced plasma insulin concentrations such that T cows had insulin concentrations 2.9- to 6.0-fold those of C cows between d 2 and 9. Dexamethasone decreased IGF-I and -II concentrations from d 5 onward. Concentrations of plasma leptin and the various IGF binding proteins were not affected by dexamethasone. Total number of follicles (> or = 5 mm) and plasma estradiol concentrations were less in T than in C cows on d 0, 1, and 4. The growth rate of the dominant follicles and maximum diameter of the dominant and subordinate follicles were not affected by dexamethasone. The diameter of the CL was 21 to 39% larger in T than in C cows between d 6 and 10. Treatment x day interaction influenced plasma cholesterol concentrations such that cholesterol levels decreased 46.8% in T cows and 19.5% in C cows between d 0 and 10. Plasma glucose concentrations were greater in T than in C cows between d 1 and 10. In summary, dexamethasone had significant effects on metabolism without a major impact on growth of the first-wave dominant follicle. Dexamethasone-induced suppression of luteal function was associated with decreased plasma IGF-I and -II concentrations.     

卵清蛋白肽对小鼠机体的免疫增强作用

对蛋白酶水解鸡卵清蛋白获得的肽类物质的理化特性及对免疫功能的调节作用进行研究。方法为凯氏定氮法测定蛋白质含量。多肽含量测定,首先经三氯醋酸沉淀样品中大分子蛋白质,按凯氏定氮法测定上清液的多肽含量。HPLC色谱法测定肽的分子量范围。免疫功能检查包括细胞免疫和体液免疫两项功能测定。与实验对照组比较,迟发型变态反应实验、小鼠半数溶血值测定实验、小鼠巨噬细胞吞噬鸡红细胞实验及NK细胞活性测定实验均有显著增强作用。提示卵清蛋白肽对实验小鼠的机体具有免疫增强作用。     

Relationships among metabolites influencing ovarian function in the dairy cow

Time series analysis was used to evaluate relationships between the uptake of metabolites, ovarian blood flow, arterial LH concentrations, and the output of steroid hormones by the ovary. There were no significant correlations between cholesterol uptake and progesterone output; therefore, cholesterol uptake was not a factor that immediately limited progesterone output. Although no significant correlations were found between cholesterol and oxygen uptake, significant cross-correlations between the uptake of glucose and oxygen by the ovary at lag 0 indicated some immediate oxidation of glucose. This result strongly indicates that glucose is a major source of energy for the bovine ovary. Arterial LH concentrations had little influence on ovarian metabolism. The examination of interrelationships among factors that influence ovarian function was useful in identifying factors that can limit ovarian activity.     

Impaired hepatic autophagic activity in dairy cows with severe fatty liver is associated with inflammation and reduced liver function

The ability of liver to respond to changes in nutrient availability is essential for the maintenance of metabolic homeostasis. Autophagy encompasses mechanisms of cell survival, including capturing, degrading, and recycling of intracellular proteins and organelles in lysosomes. During negative nutrient status, autophagy provides substrates to sustain cellular metabolism and hence, tissue function. Severe negative energy balance in dairy cows is associated with fatty liver. The aim of this study was to investigate the hepatic autophagy status in dairy cows with severe fatty liver and to determine associations with biomarkers of liver function and inflammation. Liver and blood samples were collected from multiparous cows diagnosed as clinically healthy (n = 15) or with severe fatty liver (n = 15) at 3 to 9 d in milk. Liver tissue was biopsied by needle puncture, and serum samples were collected on 3 consecutive days via jugular venipuncture. Concentrations of free fatty acids and β-hydroxybutyrate were greater in cows with severe fatty liver. Milk production, dry matter intake, and concentration of glucose were all lower in cows with severe fatty liver. Activities of serum aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and γ-glutamyl transferase were all greater in cows with severe fatty liver. Serum concentrations of haptoglobin and serum amyloid A were also markedly greater in cows with severe fatty liver. The mRNA expression of autophagosome formation-related gene ULK1 was lower in the liver of dairy cows with severe fatty liver. However, the expression of other autophagosome formation-related genes, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 3, ATG5, and ATG12, did not differ. More important, ubiquitinated proteins, protein expression of sequestosome-1 (SQSTM1, also called p62), and microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3)-II was greater in cows with severe fatty liver. Transmission electron microscopy revealed an increased number of autophagosomes in the liver of dairy cows with severe fatty liver. Taken together, these results indicate that excessive lipid infiltration of the liver impairs autophagic activity that may lead to cellular damage and inflammation.     

乌贼墨多糖对环磷酰胺致小鼠卵巢损伤的保护作用

研究乌贼墨多糖对注射环磷酰胺所致小鼠卵巢功能损伤的保护作用。本研究用雌性昆明小鼠作为模型动物,以乌贼墨多糖作为卵巢保护剂进行灌胃,腹腔注射环磷酰胺造模,检测实验小鼠的体重、卵巢指数、血清中性激素水平以及卵巢的抗氧化指标,对卵巢进行组织病理学检测并统计各级生长卵泡数量。结果显示,乌贼墨多糖能够明显缓解环磷酰胺对卵巢的毒性损伤,降低血清中FSH水平（p<0.05）和LH水平（p<0.01）,升高血清中E2水平（p<0.01）,提高卵巢组织中SOD活力（p<0.05）、GSH-Px活力（p<0.01）以及GSH含量（p<0.01）,降低卵巢中MDA含量（p<0.01）,显著增加原始卵泡数、初级卵泡数、成熟卵泡数以及卵泡总数（p<0.01）。由此表明,乌贼墨多糖可明显抑制环磷酰胺造成的卵巢损伤,保护并修复卵巢的组织结构,为其开发成临床化疗的辅助药物提供理论依据。      

汉方茶对小鼠免疫功能增强作用

目的:探讨汉方茶对小鼠免疫功能的影响。方法:192只BALB/C雄性小鼠随机分为4组,即低、中、高剂量组和溶剂对照组,前3组分别给予0.75、1.50、4.50g/kg的汉方茶,溶剂对照组给予蒸馏水,连续灌胃30d后分为4个亚组,分别检测以下指标:脏器/体质量比值、24h足跖肿胀度、脾淋巴细胞增殖能力、溶血空斑数、半数溶血值、碳廓清能力、鸡红细胞吞噬率与吞噬指数及NK细胞活性。结果:1.50、4.50g/kg剂量汉方茶可增强小鼠脾淋巴细胞增殖能力,使小鼠24h足跖肿胀度增加,促进抗体和血清凝血素的生成,提高NK细胞活性(P<0.05);各剂量组对免疫器官/体质量比值、小鼠碳廓清能力和小鼠腹腔巨噬细胞吞噬鸡红细胞能力均无明显影响(P>0.05)。结论:汉方茶对正常小鼠免疫功能具有调节作用。     

Leydig cell function in mice lacking connexin43

Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17beta-hydroxysteroid dehydrogenase (17betaHSD), 5alpha-reductase (5alphaR), and 3alpha-hydroxysteroid dehydrogenase (3alpha HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17betaHSD and 5alphaR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.     

Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice

The epithelium of the epididymal tubule has different biological functions in different regions of the tubule. Each region is further organized into lobules or intra-regional segments surrounded by connective tissue septa (CTS). Epididymal segmentation has received little direct attention, yet there is considerable evidence that expression of mRNA and protein often begins or ends precisely at the CTS border of a segment. How such 'on-off' regulation occurs coincident with the passing of the tubule from one segment to the next is unknown. This study examined the segmentation of epididymides in rats and mice. The average adult Sprague-Dawley rat and C57BL/6 mouse caput, corpus and cauda epididymides has seven, two and four, and three, one and two segments, respectively. The apoptosis response of the caput epididymal epithelium to deprivation of lumicrine factors 24 h after efferent duct ligation in rats and the epididymal expression of a marker protein, beta-galactosidase, in mice were segmented precisely. This validated both at a general response and at a specific protein level that many epididymal functions are regulated within segments. Blue dextran (molecular weight 20000) and erythrocine red (molecular weight 880) dyes infused into the interstitial space of specific segments by micropuncture were retained by the CTS of the segments. In similar micropuncture experiments, [(3)H]H(2)O (molecular weight 18) was able to diffuse into an adjacent segment relatively freely whereas [(14)C]polyethylene glycol (molecular weight 4000) could not. These studies indicate that the interstitium of intra-regional segments is organized into different physiological compartments and that these compartments play a role in regulating the epididymal epithelium.     

薏苡仁油对小鼠免疫功能影响的研究

探讨薏苡仁油对小鼠免疫功能的影响。分别以低、中、高剂量薏苡仁油(0.17、0.33、1.00 g/kg)和食用植物油连续经口灌胃ICR小鼠30 d,测定胸腺指数与脾指数,并进行脾淋巴细胞转化实验、迟发性变态反应实验、血清溶血素实验和抗体生成细胞检测、碳廓清实验、腹腔巨噬细胞吞噬鸡红细胞实验和NK细胞活性实验。结果表明:高剂量(1.00 g/kg)组能显著增强脾淋巴细胞增殖能力(以刀豆蛋白A(ConA)诱导)和迟发性变态反应(以二硝基氟苯(DNFB)诱导),升高小鼠血清溶血素水平,增强小鼠抗体生成能力和NK细胞活性。证明薏苡仁油具有增强免疫力作用。