NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary

Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.     

Soluble forms of YlCrh1p and YlCrh2p, cell wall proteins of Yarrowia lipolytica, have beta-1,3-glycosidase activity

Crh1p and Crh2p of Saccharomyces cerevisiae are cell wall proteins covalently attached to cell wall glucan and are thought to be putative glycosidases involved in cell wall remodelling. We investigated whether YlCrh1p and YlCrh2p, the Yarrowia lipolytica proteins homologous to ScCrh1p and ScCrh2p, had the required glycosidase activity for cell wall biosynthesis and maintenance. Ylcrh1Delta and Ylcrh2Delta mutants showed sensitivity to compounds that interfere with cell wall construction. Soluble forms of YlCrh1p and YlCrh2p that lacked the C-terminal consensus sequence for GPI anchoring showed glycosidase activity on laminarin, a substrate carrying beta-1,3-glycosidic linkage. Our study suggests that the YlCrh1p and YlCrh2p may participate in cell wall biosynthesis and remodelling through their beta-1,3-glycosidase activity.     

Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes

With the aim of investigating the effects of oocyte genotype and activating stimulus on the timing of nuclear events after activation, oocytes collected from hybrid B6D2F1, inbred C57BL/6 and outbred CF-1 and immunodeficient nude (NU/+) females were activated using ethanol or strontium and fixed at various time-points. Meiotic status, spindle rotation and second polar body (PB2) extrusion were monitored by fluorescence microscopy using DNA-, microtubule- and microfilament-selective probes. Although activation efficiency was similar in all groups of oocytes, a significant percentage of CF-1 and NU/+ oocytes treated with ethanol and of C57BL/6 oocytes treated either with ethanol or strontium failed to complete activation and became arrested at a new metaphase stage (MIII) after PB2 extrusion. C57BL/6 oocytes also showed slower release from MII arrest but faster progression to telophase (TII) after ethanol exposure, and they exhibited the most rapid exit from TII under both activation treatments. Strontium caused delayed meiotic resumption, spindle rotation and PB2 extrusion, but rapid TII exit, in B6D2F1, CF-1 and NU/+ oocytes when compared with ethanol. Compared with all other strains, NU/+ oocytes were significantly slower in completing spindle rotation and PB2 extrusion, irrespective of the activating stimulus, and a significant decrease in activation rates and pace of meiotic progression was observed after strontium exposure. Thus, our findings demonstrated that the kinetics of meiosis resumption and completion, spindle rotation and PB2 extrusion following parthenogenetic activation depends on both genotype-specific factors and on the activation treatment applied.     

The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse

There is some evidence suggesting that Ca2+ is involved in processes that occur during the development and function of spermatozoa. Calcium-dependent proteins, such as calmodulin, are expressed during mammalian spermatogenesis further suggesting that Ca2+ takes part in its regulation. However, the precise roles of Ca2+ in spermatogenesis remain to be elucidated. Calpains are a family of Ca(2+)-dependent cysteine proteases whose members are expressed ubiquitously or in a tissue-specific manner. Calpain has been demonstrated to mediate specific Ca(2+)-dependent processes including cell fusion, mitosis and meiosis. We herein followed the expression pattern of calpain's ubiquitous isoforms, 1 and 2, throughout spermatogenesis at the RNA and protein levels by RT-PCR and Western blotting analysis. Both RNA and protein studies revealed that these isoforms are expressed in all spermatogenic cells. The expression of calpain 1 levels is slightly higher in spermatocytes entering the meiotic phase. Both calpain isoforms are also expressed in mouse spermatozoa and are localized to the acrosomal cap. Inducing capacitated spermatozoa to undergo the acrosome reaction in the presence of a selective calpain inhibitor significantly reduced the acrosome reaction rate in a dose-dependent manner. Thus, calpain, a pluripotential protease with numerous substrates, may serve as an effector in more than one pathway in the complex process of spermatogenesis and in the events preceding fertilization, such as the acrosome reaction.     

Different milk feeding intensities during the first 4 weeks of rearing dairy calves: Part 2: Effects on the metabolic and endocrine status during calfhood and around the first lactation

Feeding dairy calves at high intensity has been demonstrated to increase milk yield in later life. We investigated the effect of 3 different feeding regimens in the preweaning period on the metabolic and endocrine status during calfhood and in heifers at the onset of the first lactation. In trial 1, 57 German Holstein calves were allocated to 3 different feeding groups: milk replacer restricted to 6.78 kg/calf per day, 11.5% solids (MR-res, n = 20), milk replacer 13.8% solids, ad libitum (MR-ad lib, n = 17), and whole milk ad libitum (WM-ad lib, n = 20). All calves received ad libitum colostrum for 3 d postnatal (p.n.). From d 4 to 27, all calves were fed according to their respective feeding regimen, resulting in average intakes of 6.38, 9.25, and 9.47 kg/d in MR-res, MR-ad lib, and WM-ad lib, respectively. Thereafter, all calves were fed according to the MR-res regimen until weaning at d 55 (gradually until d 69 p.n.). Blood samples were collected on d 0 before colostrum intake and on d 1, 3, 11, 22, 34, 43, 52, 70, 90, and 108 p.n. Liver biopsies were taken on d 19 and 100, and on d 22, 52, and 108 p.n. intravenous glucose tolerance tests were performed. The male calves (n = 8 to 10 per group) underwent also an insulin tolerance test on d 24, 54, and 110 p.n. The females (n = 28) from trial 1 were further reared and bred as common practice, and were enrolled in trial 2 when beginning the last trimester of pregnancy. Blood samples were collected monthly antepartum starting 91 d before calving and weekly (0–70 d) postpartum. Trial 1 was subdivided into 4 phases (P): P0 (d 0–1), P1 (d 2–27), P2 (d 28–69), and P3 (d 70–110 p.n.). In trial 1, the leptin and adiponectin concentrations increased with colostrum intake. Differences in fatty acids, insulin, adiponectin, revised quantitative insulin sensitivity check index (RQUICKI), and variables from the glucose tolerance tests were largely limited to P1. The MR-res group had greater RQUICKI and fatty acid values, and lower insulin and, as a trend, adiponectin concentrations than in 1 or both ad lib groups. These differences were partly sustained in P2 (fatty acids, adiponectin, and RQUICKI) and in P3 (adiponectin). The hepatic mRNA abundance of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and pyruvatcarboxylase increased from d 19 to 100. None of the blood variables were different between the groups when tested in pregnancy and lactation. Our results do not support a sustained deflection of metabolic regulation by rearing at different feeding intensities; nevertheless, the differences observed during rearing might influence nutrient utilization in later life or the cellular development of organs, such as the mammary gland, and thereby affect milk yield. Further studies involving greater animal numbers and, thus, improved power will help to sort out the mechanisms of programming body function in later life via nutrition in early life.     

Different milk feeding intensities during the first 4 weeks of rearing in dairy calves: Part 1: Effects on performance and production from birth over the first lactation

We aimed to test the effects of ad libitum feeding of whole milk (WM) or milk replacer (MR) versus restrictive feeding of MR during the first 4 wk of life on growth performance and on milk yield in the first lactation. We studied 57 German Holstein calves (29 females, 28 males) from birth until d 110 of life (trial 1). The 28 females from trial 1 were further studied during their first lactation (trial 2). In trial 1, all calves were randomly allocated at birth to 1 of 3 groups: MR-res [n = 20, 6.78 kg MR (11.5% solids)/calf per day], MR-ad lib (n = 17, 13.8% solids) or WM-ad lib (n = 20). All calves received colostrum ad libitum from their dam until d 3 of age. From d 4 to 27, calves were fed according to their group regimen. From d 28 to 55, all calves received MR-res feeding and were then gradually weaned until d 69. We recorded body weight (until d 110) and feed intake (amount, metabolizable energy, and frequency of liquid feed intake until weaning). We estimated the profitability of the different feeding regimens, taking into account income from milk yield (trial 2) and feed costs during rearing. In trial 1, the calves from WM-ad lib and MR-ad lib had total metabolizable energy intakes 2.02- and 1.65-fold greater than the MR-res group during the first 4 wk of life. During this period, concentrate intake did not differ among groups, but tended to be greater in WM-ad lib than in MR-ad lib calves from d 28 to 69. The MR-res calves visited the automatic feeders more often than the ad libitum-fed groups during differential feeding, but 70% of the visits were unrewarded (<10% in the ad libitum-fed calves). When all calves were fed at the MR-res level, the average proportion of unrewarded visits was 65% in all groups. Average daily gain and body weight were greater among MR-ad lib and WM-ad lib calves than among MR-res animals during the first 4 wk of life, but not from d 1 to 110. In trial 2, age at first calving, dry matter intake, and body weight over the first 10 mo of lactation were not different among groups, nor was milk composition. Milk yields (305 d) were numerically but not statistically greater in the ad libitum-fed groups during the first lactation (+765 kg for WM-ad lib vs. MR-res; +612 kg for MR-ad lib vs. MR-res). Feeding WM-ad lib and MR-ad lib was 1.37- and 1.21-fold more costly than MR-res, respectively, but amounted to 18, 15, and 13% of the total estimated feed costs until first calving in WM-ad lib, MR-ad lib, and MR-res, respectively. Our study confirms that ad libitum feeding is an attractive measure for rearing dairy calves, both for animal welfare and—with the caveat of a small sample size in trial 2 that led to insufficient power—economic profit from milk.     

Characterization of mouse sperm TMEM190, a small transmembrane protein with the trefoil domain: evidence for co-localization with IZUMO1 and complex formation with other sperm proteins

TMEM190, a small transmembrane protein containing the trefoil domain, was previously identified by our proteomic analysis of mouse sperm. Two structural features of TMEM190, 'trefoil domain' and 'small transmembrane protein', led us to hypothesize that this protein forms a protein-protein complex required during fertilization, and we characterized TMEM190 by biochemical, cytological, and genetic approaches. We showed in this study that the mouse Tmem190 gene exhibits testis-specific mRNA expression and that the encoded RNA is translated into a 19-kDa protein found in both testicular germ cells and cauda epididymal sperm. Treatment of the cell surface with proteinase K, subcellular fractionation, and immunofluorescence assay all revealed that mouse TMEM190 is an inner-acrosomal membrane protein of cauda epididymal sperm. During the acrosome reaction, TMEM190 partly relocated onto the surface of the equatorial segment, on which sperm-oocyte fusion occurs. Moreover, TMEM190 and IZUMO1, which is an immunoglobulin-like protein required for gamete fusion, co-localized in mouse sperm both before and after the acrosome reaction. However, immunoprecipitates of TMEM190 contained several sperm proteins, but did not include IZUMO1. These findings suggest that a mouse sperm protein complex(es) including TMEM190 plays an indirect role(s) in sperm-oocyte fusion. The role(s), if any, is probably dispensable since Tmem190-null male mice were normally fertile.     

Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to -7.0 ° C, induced to freeze externally, and finally cooled at 20 ° C/min to -70 ° C. IIF that occurred during the 20 ° C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were -34, -40, -35, and -25 ° C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10-15 ° C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-stranded Aqp3 RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.     

Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation,granulosa cell proliferation,steroid hormone and PGE2 release in cultured rat ovarian follicles

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.     

四平针2+1罗纹的减针留边工艺研究

通过分析四平针2+1罗纹和相对2+2罗纹的组织结构和外观特点,绘制编织意匠图和结构图.在四平针2+1罗纹组织结构基础上,详细介绍两种减针留边工艺,包括四平针2+1罗纹的减针留边和相对2+2罗纹的减针留边,并在双针床电脑横机上编织羊毛衫实物.结果表明:四平针2+1罗纹组织结构的两种减针留边工艺均能满足产品设计需求,确保产...     

Effect of polymorphisms in the leptin, leptin receptor, and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes and genetic polymorphism of milk proteins on cheese characteristics

Cheese production has increased worldwide during the last decade and is expected to increase within the coming decade as well. Despite this, the relations between cow genetics and cheese characteristics are not fully known. The aim of this study was to determine if polymorphisms in the leptin (LEP), leptin receptor (LEPR), and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes as well as genetic variants of β-casein (β-CN), κ-CN, and β-lactoglobulin (β-LG) affect technological properties important for cheese production and, hence, could act as genetic makers for cheese quality. Individual milk samples from the Swedish Red and the Swedish Holstein breeds were analyzed for sizes of CN micelles and fat globules as well as rennet-induced gel strength, gelation time, and yield stress. Model cheeses were produced to study yield, hardness, and pH of the cheeses. The A1457G, A252T, A59V, and C963T single nucleotide polymorphisms (SNP) were analyzed on the LEP gene, the T945M SNP on the LEPR gene, and the Nt984+8(A-G) SNP on the DGAT1 gene. In addition, genetic variants of β-CN, κ-CN, and β-LG were determined. The results indicate that technological properties were influenced by the LEPR_T945M polymorphism, which had an association with gel strength, yield stress, and cheese hardness (T > C). However, also LEP_A252T was shown to affect gel strength (T > A), whereas the LEP_A59V had an effect on fat globule size (T > C). For the milk protein genes, favorable effects were found for the A and B variants of β-LG and κ-CN, respectively, on gel strength, gelation time, and yield stress. In addition, the B variant of κ-CN was shown to be associated with smaller CN micelles than the A variant. Thus, the results demonstrate potential genetic markers for cheese characteristics. However, milk composition traits also affected the obtained results, thus making it necessary to thoroughly assess the different aspects regarding the influence of gene effects on cheese characteristics before directly selecting for certain alleles or genetic variants to improve the processing and quality of cheese.     

尿苷二磷酸-葡萄糖醛酸转移酶基因UGT2B7和UGT2C1与赤拟谷盗磷化氢抗性关系研究

为明确尿苷二磷酸-葡萄糖醛酸转移酶基因UGT2B7和UGT2C1基因与赤拟谷盗Tribolium castaneum磷化氢(PH3)抗性之间的关系.本研究以PH3敏感和抗性种群的赤拟谷盗为研究对象,采用实时荧光定量PCR(qPCR)和RNA干扰技术,解析UGT2B7和UGT2 C1基因的表达模式,以及基因沉默后赤拟谷盗...     

The α-amylase, α-amylase inhibitor and proteinase inhibitor activities of proteins extracted from wheat varieties and their influence on the baking quality

The grain of six winter and spring wheat varieties, harvested in 1987, differing in baking quality were included in the study. A number of technological analysis were performed. Albumins, globulins and gliadins were washed out. The location of inhibitory activities in extracted proteins, against α-amylases of various origin. were tested and compared to the baking quality of the wheat grain. Also, the location of antitryptic activity and endogenous amylase activity in extractable proteins was compared to baking quality factors. Among the studied inhibitory activities against mammalian α-amylases some correlation were found between the inhibition activity of hog pancreas α-amylase and sedimentation test (r = 0.88). Furthermore inhibitory activities of wheat proteins towards insects α-amylase shown significant correlation between the inhibitors of Anagasta kuehniella α-amylase and sedimentation test (r = 0.90). as well as crude protein content (r = 0.86). Also the inhibition activity against Sitophilus granarius α-amylase versus sedimentation test presented some correlation (r = 0.80). Endogenous amylase activities (α- and β-amylase) have an inversely proportional correlation to crude protein content (r = ?0.82) and sedimentation test (r = ?0.85).     

Randomized clinical trial of the effect of a fixed or increasing milk allowance in the first 2 weeks of life on health and performance of dairy calves

The objective of this study was to describe the effect of offering a fixed or increasing milk allowance in the first 1 to 2 wk of life. We hypothesized that calves offered a fixed amount of milk early in life would not experience more scours, but rather would experience improved health and growth compared with calves that had their daily milk allowance slowly increased over a period of 1 to 2 wk. This randomized controlled clinical trial was conducted on 5 dairy farms in Minnesota with both a summer (June–August 2016) and winter (December–February 2017) period of enrollment. Heifer calves were enrolled at birth, weighed, and systematically assigned by birth order to either the slowly increasing (INC) control group or fixed allowance (FIX) treatment group by farm personnel. Calves assigned to the INC group were slowly increased from 4 to 5 L/d to gradually reach the full peak milk allowance of 6 to 8 L/d over a 7- to 14-d period, whereas calves assigned to the FIX group were offered a full peak milk allowance of 6 to 8 L/d beginning on d 1 after birth. The average FIX calf consumed an extra 14 L of milk as compared with INC calves over the first 2 wk of life, corresponding to an average INC intake of 5 L/d during first 1 to 2 wk of life as compared with an average intake of 6.8 L/d in FIX calves. Study technicians visited all farms weekly to collect health and performance data. Multivariable mixed models were used to describe the effect of treatment (INC/FIX) on 3-wk average daily gain (kg/d), 3-wk weight (kg), and hip height at wk 1, 3, and 7, controlling for the effect of season, birth weight, and the random effect of calf within farm. Multivariable logistic regression models were used to describe the effect of treatment on odds of technician and producer reported health events. A total of 1,264 heifer calves were enrolled (FIX n = 641; INC n = 623) with no difference in enrollment weight or hip height between groups. By 3 wk of age, FIX calves weighed 1.4 (0.59) kg more than INC calves, though the magnitude of this difference varied depending on the period of time INC calves were slowly increased in milk allowance (7 vs. 10 vs. 14 d). Calves in the FIX group grew 0.1 kg/d faster and were taller at wk 3 (0.3 ± 0.15 cm) of life. Forty-two percent (536/1,264) of all enrolled calves had a first treatment event, with no effect of treatment on technician-reported health scores and no overall effect on producer-reported treatment or mortality events. Under the conditions of this study, offering a fixed milk allowance from d 1 of life improved calf growth during the first 3 wk as compared with a gradual increase in milk allowance, with no detrimental effect on calf health.