Multiple vitellogenins from the Haemaphysalis longicornis tick are crucial for ovarian development

Ovarian development and egg maturation are crucial processes for the success of reproduction in ticks. Three full-length cDNAs encoding the precursor of major yolk protein, vitellogenin, were obtained from cDNA libraries of the Haemaphysalis longicornis tick and designated as HlVg-1, HlVg-2 and HlVg-3. The HlVg mRNAs were found in fed females with major expression sites in the midgut, fat body and ovary. Native PAGE and Western blot demonstrated that HlVgs in the hemolymph, fat body and ovary of fed females consisted of four major polypeptides. RNAi results showed that HlVg dsRNA-injected ticks obtained lower body weight, egg weight and showed higher mortality of engorged females after blood sucking than control groups. Our results indicate that all HlVgs are essential for egg development and oviposition.     

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.     

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h) promoted optimal parthenogenetic activation.     

Resistance in cattle to Haemaphysalis (Kaiseriana) longicornis

Samples of each instar of the 3-host tick, Haemaphysalis (Kaiseriana) longicornis were collected from grazing Bos taurus cattle to investigate their survival on hosts with previous exposure to the tick and on previously unexposed ‘naive’ hosts. The ticks were allocated to categories based on their size to observe at what stage of development mortality occurred. Analysis of the age structure of the ticks showed that small but variable proportions of larvae, nymphs and females matured on previously exposed hosts and larger proportions on the naive hosts. This suggests that cattle acquire and express different levels of resistance against each instar of the tick. The technique used has potential for estimating levels of resistance of hosts to 3-host ticks under field conditions.     

Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k_cat / K_m = 0.19 and 0.0023 M^− 1 S^− 1, respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.     

Morphological characterization of the ovary and oocytes vitellogenesis of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

This study presents the morphology of the ovary, as well as the process of the vitellogenesis in oocytes of the tick Rhipicephalus sanguineus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a wall formed by small epithelial cells with rounded nuclei which delimit the lumen. The oocytes in the different developmental stages in this tick species were classified into five stages (I-V). They remain attached to the ovary during vitellogenesis by a cellular pedicel and afterwards the mature oocytes (stage V) are released into the ovary lumen.     

Effect of ovary preservation period on recovery rate and categories of dromedary camel oocytes

The aim of this study was to evaluate the effect of different periods of ovary preservation at 25–30 °C for 5, 6, 7, 9, 12 and 24 h on recovery rate and oocyte categories of dromedary camel oocytes. Camel ovaries were collected from El-Bassatein slaughterhouse, Cairo. The collected ovaries were placed immediately after slaughtering into thermos in saline solution (0.9% NaCl) supplemented with antibiotics (100 IU penicillin and 100 μg streptomycin/ml) at 25–30 °C and transported to the laboratory within 4–5 h. Ovaries were washed three times with warmed (30 °C) phosphate buffer solution (PBS) and one time with ethanol (70%). All visible follicles on the ovarian surface (2–8 mm in diameter) were counted. Oocytes were aspirated using a 20-gauge hypodermic needle. Oocyte yield was recorded and the number of oocytes/ovary was calculated. Oocytes were classified into five categories (compact, partial denuded, denuded, shrunken and cleaved oocytes). Results show that average number of follicles on each ovary was not significantly affected by preservation period, although tended to reduce only after 5 h of ovary preservation. However, this number was insignificantly reduced by increasing period of ovary preservation more than 5 up to 24 h. Average number of oocytes on each ovary was significantly (P < 0.05) reduced only between 5 and 6 h of ovary preservation. Average number of oocytes showed higher reduction rate between 5 and 6 h from 12.4 to 9.3/ovary as well as between 9 and 12 h. Oocyte recovery rate showed insignificant decrease from 88.1% at 5 h to 78.6% at 9 h of preservation. However, it showed significant (P < 0.05) reduction to 62.0% between 9 and 12 h, then insignificantly decreased to 58.6 at 24 h of preservation of the ovaries. Frequency distribution and recovery rate of each category was the highest for compact oocytes and the lowest for cleaved oocytes at all periods of preservation. Increasing preservation period significantly (P < 0.05) decreased frequency distribution of compact and cleaved oocytes, while increased frequency distribution of partial denuded, denude and shrunken oocytes.It might be concluded from the present results that the preservation of dromedary camel ovaries at 25–30 °C for 5–6 h was effective for maintaining the oocytes quality and recovery rate compared with the other preservation periods.     

猪卵巢体外保存温度对卵母细胞染色质构型和成熟能力的影响

Most porcine oocytes used in studies on embryo biotechnology and the in vitro production of embryos are currently obtained from the ovaries of slaughtered gilts. The duration and temperature during ovary transportation and handling might, therefore, affect the recovery of culturable COCs, chromatin configuration and developmental competence of oocytes. The effects of ovary storage temperature on chromatin configuration and in vitro maturation of porcine oocytes were examined in this study. Ovaries collected from a slaughterhouse were stored in vitro for 8 h under different temperatures. The results showed that more culturable COCs were isolated from the ovares stored at 39℃ than that from ovaries stored at 31℃ or 20℃ and before storage. Thirty-one centidegree was the best storage temperature in terms of cumulus expansion, nuclear maturation and morphology of the first polar body after in vitro maturation culture. The ability of cumulus expansion was completely lost in COCs derived from ovaries stored at 39℃ for 8 hours. Ovary storage （at both 31℃ and at 20℃ ） increased the proportion of oocytes with the GVc configuration in which chromatin condensed into a single big clump at the nucleolus and the functional significance of this configuration needs further investigations [ Acta Zoologica Sinica 51 （5）： 919 923, 2005].     

Potential applications of sheep oocytes as affected by vitrification and in vitro aging

The present study was carried out to investigate how the interactions between aging, vitrification and post-warming interval affect the credibility of sheep MII-oocytes for in vitro fertilization (IVF), intracytoplasmic injection (ICSI), and parthenogenetic activation (PA). According to our results, aged oocytes had significantly higher rates of chromosome and spindle abnormalities compared to young oocytes. However after vitrification-warming, the total rates of these abnormalities were not significantly different between aged and young oocytes. Unvitrified-aged, and vitrified young and aged oocytes had comparable ultrastructural characteristics, whereas they were completely dissimilar in compared with unvitrified-young oocytes. Although mRNA abundance was reduced during vitrification-warming in both aged and young oocytes, the post-warming interval could improve the relative mRNA abundance. Aged oocytes had lower capacity for IVF and ICSI in compared with young oocytes, but had similar pattern for PA process. The vitrification process decreased developmental competence of both aged and young oocytes in compared with young ones, particularly when warmed oocytes were rested for 2 h before IVF, ICSI and PA. The results of the present study showed that in vitro aged oocytes had higher capacity to be used for parthenogenetic studies rather than IVF and ICSI. Furthermore, it was shown that vitrified oocytes had a time-dependent decline in quality and developmental potential. Notably, the speed of this decline was higher in vitrified-young oocytes, indicating that the vitrified oocytes do not require to be rested post warming. Conclusively, the results of this study can be useful in preserving in vitro aged oocytes to provide a valuable and easy access source of oocytes for research purposed studies.     

Analysis of the chromosome complement of frozen-thawed mouse oocytes after parthenogenetic activation

Frozen-thawed mouse oocytes were artificially activated with Sr²⁺ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen-thawed and freshly collected oocytes and the majority of oocytes (>90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozenthawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0°C or 37°C for extended periods beyond those required for protection. The frequency of aneuploidy was similar for frozen-thawed and fresh oocytes but oocytes held at 0°C without DMSO or held at 37°C with DMSO for 1 hr showed a 2.5 and 12-fold increase in the frequency of aneuploidy compared with oocytes subjected to a conventional oocyte/embryo freezing regime. It is concluded that the procedures used in successful oocyte cryopreservation do not increase the incidence of chromosomal abnormalities of maternal origin in the resulting embryos. © 1995 wiley-Liss, Inc.     

Intercellular bridges between oocytes in the chicken ovary

Summary Fine structural analysis of the functional (left) ovary of the newly-hatched chick reveals the presence of true intercellular bridges between developing oocytes in the early stages of the meiotic prophase. These structures are characterized by: 1) cytoplasmic continuity between the participating oocytes, 2) a dense, fibrillar material beneath the lateral limiting membrane and 3) numerous cellular organelles within their confines. In addition, microtubular elements, parallel to the long axis of the bridge, are routinely observed. This latter finding suggests that intercellular bridges originate through incomplete cytokinesis of mitotically active oogonia and that the dense material beneath the limiting membrane may represent the cortical microfilaments associated with the contractile ring. Functionally, these structures may serve as channels for transfer of nutrients and organelles between oocytes although the possibility that certain oocytes function as nurse cells, in the sense that these cells exist in invertebrate ovaries, seems unlikely. In addition, intercellular bridges may be responsible for both restriction of oogonial mitoses and meiotic synchrony.Partial support for this study was provided by grant DE-00241 from the National Institute for Dental Research administered by Dr. Melvin Hess. We gratefully acknowledge his support of the initial aspects of this study. We are pleased to express our appreciation to Mrs. Cindy G. Wilcox, Mr. Lloyd Thibodeau and Mr. Don Driscoll for their expert technical assistance.     

Trichloroethylene metabolism in the rat ovary reduces oocyte fertilizability

Exposure to trichloroethylene (TCE, an environmental toxicant) reduced oocyte fertilizability in the rat. In vivo, TCE may be metabolized by cytochrome P450 dependent oxidation or glutathione conjugation in the liver or kidneys, respectively. Cytochrome P450 dependent oxidation is the higher affinity pathway. The primary isoform of cytochrome P450 to metabolize TCE in the liver, cytochrome P450 2E1, is present in the rodent ovary. Ovarian metabolism of TCE by the oxidative pathway and the production of reactive oxygen species (ROS) may occur given the presence of the metabolizing enzyme. The objectives of this study were to define the sensitive interval of oocyte growth to TCE exposure, and to determine if TCE exposure resulted in the formation of ovarian protein carbonyls, an indicator of oxidative damage. Rats were exposed to TCE in drinking water (0.45% TCE (v/v) in 3% Tween) or 3% Tween (vehicle control) during three 4-5 day intervals of oocyte development preceding ovulation. Oocytes from TCE-exposed females were less fertilizable compared with vehicle-control oocytes. Immunohistochemical labeling of ovaries and Western blotting of ovarian proteins demonstrated TCE treatment induced a greater incidence of protein carbonyls compared with vehicle controls. Protein carbonyl formation in the ovary is consistent with TCE metabolism by the cytochrome P450 pathway. Oxidative damage following ovarian TCE metabolism or the presence of TCE metabolites may contribute to reduced oocyte fertilizability. In summary, these results indicate maturing oocytes are susceptible to very short in vivo exposures to TCE.     

Ultrastructural detection of proteins, lipids and carbohydrates in oocytes of Amblyomma triste (Koch, 1844) (Acari; Ixodidae) during the vitellogenesis process

The ovary of the tick Amblyomma triste is classified as panoistic, which is characterized by the presence of oogonia without nurse and follicular cells. The present study has demonstrated that the oocytes in all developmental stages (I-IV) are attached to the ovary through a pedicel, a cellular structure that synthesizes and provides carbohydrate, lipids and proteins supplies for the oocytes during the vitellogenesis process. The lipids are deposited during all oocyte stages; they are freely distributed as observed in stages II, III and IV or they form complexes with other elements. The proteins are also deposited in all stages of the oocytes, however, in lower concentration in the stage IV. There is carbohydrate deposition from oocytes in the stage II as well as in stages III and IV. In addition, the present work has demonstrated that the oocyte yolk of A. triste has a glycolipoprotein nature and the elements are deposited in the following sequence: firstly the lipids and proteins, and finally the carbohydrates.     

Azadirachta indica A. Juss (neem) induced morphological changes on oocytes of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) tick females

Many studies have been conducted with plants whose extracts have the potential to be used for pest control. One of these plants is Azadirachta indica (neem), whose main active ingredient is azadirachtin, a compound shown to have acaricide and insecticide activity. Rhipicephalus sanguineus (brown dog tick) is currently considered to be an “urban pest,” because of its high levels of infestation and its ability to attack humans. In the present study partially and fully engorged R. sanguineus females were exposed to aqueous extracts of neem at concentrations of 10% and 20%, and to a control treatment. The results showed that differently from what was observed in the control, the pedicel cells of females exposed to neem at both concentrations lost their original shape. In the latter cases, the cytoplasm of the cells became fully vacuolated, especially near the germinal vesicle (oocyte nuclei) and in the oocyte pole, which is in contact with the cells of the pedicel. Oocytes in early stages of development (I and II) of ticks treated with both concentrations had irregular germinal vesicle, including the presence of two nucleoli as well as fragments of these. Oocytes in stages IV and V of exposed individuals showed full granular cytoplasm with bigger yolk granules when compared to the early stages. Chorion of mature oocytes was also altered, showing folds and deformations along their entire extension. The observed changes in cells of the reproductive system of R. sanguineus, especially in the oocytes, indicated the potential of neem as a new alternative method to control these ectoparasites.     

TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary

Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.     

小鼠孤雌胚与体内正常胚H3K27三甲基化模式的差异

为了考察小鼠(Mus musculus)孤雌激活胚胎H3K27三甲基化模式与体内正常胚胎之间的差异,以及曲古抑菌素A(TSA)对孤雌胚H3K27三甲基化水平的影响,探究表观遗传修饰对孤雌胚胎发育的作用。首先,用H3K27me3特异性抗体对MⅡ期卵母细胞染色,利用激光共聚焦对其荧光强度进行检测,结果发现该时期的甲基化荧光强度相对较低。接着,采用同样的方法对小鼠孤雌胚胎和体内正常胚胎植入前各时期的H3K27me3模式进行比较,结果显示,从2-细胞到囊胚期孤雌组呈现逐渐升高的趋势,与体内组变化趋势完全相反,且总体平均荧光强度较体内组普遍偏低。孤雌胚胎经TSA处理后,处理组和未处理组在前三个时期虽然没有显著性差异(P0.05),但是处理之后的H3K27三甲基化水平有所提高,囊胚期与未处理组相比有显著性差异(P0.05)。以上结果表明,小鼠孤雌胚胎的H3K27三甲基化模式与体内胚胎之间存在着巨大的差异,这可能是造成孤雌胚胎发育能力差的重要原因之一。TSA处理对H3K27me3模式造成了一定的影响,使体外培养环境有所改善,这可能对提高孤雌胚胎发育能力具有一定的意义。     

The ovary structure, previtellogenic and vitellogenic stages in parthenogenetic species Dactylobiotus dispar (Murray, 1907) (Tardigrada: Eutardigrada)

The reproductive system of Dactylobiotus dispar consists of the ovary and the oviduct that opens into the rectum. The sack-like ovary is filled with the developing oocytes, which are assisted by the trophocytes. In D. dispar, the mixed vitellogenesis takes place. One part of the yolk material is produced inside the oocyte (autosynthesis), the second part is absorbed by micropinocytosis while the third part is synthesized in the trophocytes and is transported to the oocytes through the cytoplasmatic bridges. Moreover, rRNA, lipids and mitochondria are transfered from the trophocytes to the oocytes. The histochemical researches show that the reserve material accumulated in the oocytes contains proteins, polysaccharides and lipids.