Oocyte Maturation is Required for Correct Sperm Chromatin Rearrangement

Veterinary Research Communications -     

成功是选择出来的

时下许多企业、许多人都在研究如何走出困境、如何成功,小企业想做大,大企业想做成“航空母舰”,即使奄奄一息的企业也在梦想着有朝一日能够辉煌。饲料行业的变革序幕已经拉开．这是一个优胜劣汰的过程,也就是我们这里要说的“选择”过程。有哲人说：“失败的原因多种多样,成功的经验则惊人得相似”。这句话是很有道理的,这里我们就尝试通过“选择”这个角度来分析饲料行业的一些规律。[第一段]     

谷氨酰胺和IGF-I对晋岚绒山羊卵母细胞体外成熟的影响

为了探讨在成熟培养液中添加谷氨酰胺和IGF-I对晋岚绒山羊母羔卵母细胞体外成熟的影响。随机选择12只6-8周龄晋岚绒山羊母羔为试验动物,用FSH进行超数排卵,活体采集卵母细胞,并对未成熟的卵母细胞进行体外成熟培养。在培养液中添加0、50、100和150μg/mL的谷氨酰胺及0、25、50和100 ng/mL的IGF-I。结果表明：培养液中谷氨酰胺添加量为100μg/mL时,能显著提高卵母细胞成熟率（P〈0.05）;在培养液中添加100μg/mL谷氨酰胺的基础上,再添加50 ng/mL的IGF-I可以进一步显著提高卵母细胞的成熟率（P〈0.01）。     

Morphology,morphometry and chromatin distribution in llama sperm nuclei

The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 μm², perimeter 13.16 μm, length 5.12 μm, width 2.81 μm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.     

牛人工授精所需有效精子数的研究

为了探索不同数量有效精子与受胎率之间的关系,试验用200万、400万、600万、800万有效精子数冻精输配母牛。结果表明,600万与800万有效精子数受胎率比较差异不显著(P>0.05),400万和200万有效精子数受胎率显著低于800万(P<0.05)。该结果可为相关单位进一步优化细管冻精生产提供参考。     

Assessment of buffalo (Bubalus bubalis) sperm DNA fragmentation using a sperm chromatin dispersion test

Chromosomal fragmentations or damage in sperm DNA has considerable value in determination of semen quality. However, rapid and/or simple method to assess sperm DNA integrity in buffalo has apparently not been reported. In the present study, SCD was used for the first time in buffalo bulls for assessment of sperm DNA fragmentation. A modified SCD protocol, under bright field microscope was developed and validated by comparison with other routine tests which can be used for processing of samples. The DNA fragmentation index (DFI) from SCD was correlated with semen quality parameters viz. viability (r=-0.68, p<0.05), membrane integrity (r=-0.74, p<0.05) and capacitation status (r=-0.69, p<0.05). The amount of DNA fragmentation assessed by SCD was highly correlated (R=0.874, p<0.05) with results of acridine orange test (AOT), a traditional method of assessing DNA damage. There were no significant differences between two observers with regards to scoring dispersion patterns. Therefore, the SCD test can be routinely used for detection of DNA fragmentation in buffalo sperm, with potential for replacing conventional time consuming tests.     

Sperm nuclear protamines: A checkpoint to control sperm chromatin quality

Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint during sperm development rendering protamines as suitable biomarkers for the estimation of sperm quality. This review presents an overview of our current knowledge on protamines comparing gene and protein structures between different mammalian species with particular consideration given to man, mouse and stallion. At last, recent insights into the possible role of inherited sperm histones for early embryo development are provided.     

饲料企业,危机管理是门必修课

<正>从霸王洗发水的"致癌门"到云南白药的"功效门",再到丰田的"刹车门",加之以前三鹿的"结石门"、农夫山泉"砒霜门"……他们像是开了会约好一样:以最大的尺度来挑战各位人们的极限。这一切不但带给企业灭顶之灾,也让消费者极度困惑——我们还能相信什么呢?     

Oocyte maturation,embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification

Objective

To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification.

Methods

Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions.

Results

The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P < 0.05), respectively. Further, COCs in V2 group showed higher mean (±SD) percentages of cleavage compared to V1 group (31.8 ± 1.0 vs 21.7 ± 2.8%; P < 0.05). GDF9 and BMP15 expression levels were higher in COCs in the control than of the vitrification groups (P < 0.05). In addition, expression level of GDF9 and BMP15 was higher in V2 group than in V1group (P < 0.05). The expression of HAS2 and FGF17 in V1 group was lower (P < 0.05) than that of the V2 groups.

Conclusions

Expression of oocyte maturation genes was affected by vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

     

Effect of density gradient composition on in vitro maturation of stallion sperm

The present study was conducted to determine the influence of density gradient composition on in vitro capacitation of stallion spermatozoa. In Experiment I spermatozoa were isolated on either 90% Percoll (P), 90% arabinogalactan (AG), or 12% BSA gradients and then challenged with 1 μM A23187 (15, 150, and 270 min) and heat-solubilized equine zonae pellucidae (270 min, sEZP, 2 ZP/μl). The P gradient enhanced the percentage of progressively motile spermatozoa (PMS) more (P=.0001) than AG or BSA gradients immediately post-processing, but was not sustained throughout the culture period. The viability for P-separated spermatozoa was higher (P=.01) than that of BSA or AG-separated spermatozoa. Gradient composition had no effect (P=.68) on the percentage of live, acrosome reacted spermatozoa (PAR), before and following ionophore and sEZP challenge. In Experiment II, the number of spermatozoa penetrating the ZP of saltstored oocytes was not influenced (P=.35) by gradient composition; however, the number of spermatozoa bound per oocyte was higher (P=.02) for P-separated spermatozoa than for AG or BSA-separated spermatozoa. These data suggest that isolation on a P density gradient may enhance in vitro capacitation of stallion spermatozoa.     

Fatty acid content in epididymal fluid and spermatozoa during sperm maturation in dogs

Background: During sperm maturation, there is a reorganization of fatty acids from plasmatic membrane of the spermatozoa, which allows higher membrane integrity and acquisition of sperm motility. However, the fatty acid profile during sperm maturation remains unclear in dogs. Thus, the aim of this study was to identify the fatty acids from the epididymal spermatozoa and plasma during the sperm maturation, and observed changes in the motility and plasmatic membrane parameters. Twenty one adult dogs were used, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy, computer-assisted motility analysis, sperm plasma membrane permeability and the fatty acid analysis(lipids were extracted, transmethylated and analyzed by chromatography).Results: Caput and corpus sperm showed lower values for the motility variables evaluated and plasmatic membrane integrity, indicating different levels of the fatty acids organization. Saturated, monounsaturated and polyunsaturated fatty acids were in higher concentrations in the spermatozoa from epididymis cauda. Highlighting the presence of caprylic, stearic and docosahexaenoic acids.Conclusions: These findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.     

RNA and protein synthesis is required for Ancylostoma caninum larval activation

The developmentally arrested infective larva of hookworms encounters a host-specific signal during invasion that initiates the resumption of suspended developmental pathways. The resumption of development during infection is analogous to recovery from the facultative arrested dauer stage in the free-living nematode Caenorhabditis elegans. Infective larvae of the canine hookworm Ancylostoma caninum resume feeding and secrete molecules important for infection when exposed to a host mimicking signal in vitro. This activation process is a model for the initial steps of the infective process. Dauer recovery requires protein synthesis, but not RNA synthesis in C. elegans. To determine the role of RNA and protein synthesis in hookworm infection, inhibitors of RNA and protein synthesis were tested for their effect on feeding and secretion by A. caninum infective larvae. The RNA synthesis inhibitors α-amanitin and actinomycin D inhibit feeding dose-dependently, with IC(50) values of 30 and 8 μM, respectively. The protein synthesis inhibitors puromycin (IC(50)=110 μM), cycloheximide (IC(50)=50 μM), and anisomycin (IC(50)=200 μM) also displayed dose-dependent inhibition of larval feeding. Significant inhibition of feeding by α-amanitin and anisomycin occurred when the inhibitors were added before 12h of the activation process, but not if the inhibitors were added after 12h. None of the RNA or protein synthesis inhibitors prevented secretion of the activation-associated protein ASP-1, despite nearly complete inhibition of feeding. The results indicate that unlike dauer recovery in C. elegans, de novo gene expression is required for hookworm larval activation, and the critical genes are expressed within 12h of exposure to activating stimuli. However, secretion of infection-associated proteins is independent of gene expression, indicating that the proteins are pre-synthesized and stored for rapid release during the initial stages of infection. The genes that are inhibited represent a subset of those required for the transition to parasitism, and therefore represent interesting targets for further investigation. Furthermore, while dauer recovery provides a useful model for hookworm infection, the differences identified here highlight the importance of exercising caution before making generalizations about parasitic nematodes based on C. elegans biology.     

H1foo is indispensable for meiotic maturation of the mouse oocyte

Oocyte-specific linker histone H1foo is localized in the oocyte nucleus, either diffusely or bound to chromatin, during the processes of meiotic maturation and fertilization. This expression pattern suggests that H1foo plays a key role in the control of gene expression and chromatin modification during oogenesis and early embryogenesis. To reveal the function of H1foo, we microinjected antisense morpholino oligonucleotides (MO) against H1foo into mouse germinal-vesicle stage oocytes. The rate of in vitro maturation of the antisense MO group was significantly lower than that of the control group. Eggs that failed to extrude a first polar body following injection of antisense MO arrested at metaphase I. Additionally, co-injection of in vitro synthesized H1foo mRNA along with antisense MO successfully rescued expression of H1foo and improved the in vitro maturation rate. There was no difference in the rate of parthenogenesis between the antisense MO and control groups. These results indicate that H1foo is essential for maturation of germinal vesicle-stage oocytes.     

猪卵母细胞体外成熟过程中p90rsk未被MAPK激活

从屠宰场获得的初情期母猪卵巢中的卵母细胞 ,在修改后的 TCM199中进行体外成熟培养。在卵母细胞发育的一定时期取样 ,利用 SDS- PAGE蛋白质电泳和免疫印迹技术 ,研究了 MAPK(丝裂原激活蛋白激酶 ,又称 ERK)和p90 rsk (ribosomal S6 kinase)对猪卵母细胞成熟的调节。结果表明 ,修改后的 TCM199可以满足猪卵母细胞成熟的需要 ,但猪卵母细胞在体外成熟过程中 ,p90 rsk未随 MAPK激活而激活 ,说明维持猪卵母细胞的 M 阻滞与 MAPK有关 ,而与 p90 rsk无关。     

Chicken PRDX3 is required for proliferation of chicken embryo fibroblast cells

ABSTRACT

1. This experiment investigated the influence of chicken PRDX3 on cell proliferation in chick embryo fibroblast cells using PRDX3 knockdown technology.

2. A methyl thiazolyl tetrazolium (MTT) assay was performed to assess the effect of chPRDX3 knockdown on fibroblast proliferation. The antioxidant effect was investigated to determine if it directly mediated fibroblast cell proliferation.

3. To determine the role of chPRDX3 on cell proliferation, an siRNA mediated knockdown was performed in chick fibroblast cells using an in vitro assay. The proliferation of fibroblast cells transfected with siPRDX3 #3 and siPRDX3 Mix was significantly decreased after 48 h (P < 0.01). In addition, the knockdown of chicken PRDX3 suppressed cell proliferation through an increase in oxidative stress.

4. The results demonstrated that chPRDX3 is required for cell proliferation in chicken fibroblast cells. Such findings have important implications for the maintenance of chicken fibroblast cells.     

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88^−/−) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88^−/− mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88^−/− mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-γ responses by NKT, CD4⁺ and CD8⁺ T lymphocytes, and production of high levels of IL-10. MyD88^−/− mice replenished with IL-12 and IFN-γ abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-γ-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.