Morphological, histological, and ultrastructural studies of the ovary of the cattle-tick Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae)

This study presents the morphology of the ovary, as well as the dynamics of the vitellogenesis process in oocytes of the cattle-tick Boophilus microplus. 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 lumen delimitated by a wall of small epithelial cells with rounded nuclei. In this tick species, the oocytes were classified into six stages varying from I to VI and according to: cytoplasm appearance and presence of the germ vesicle, yolk granules, and chorion. Oocytes of various sizes and at different developmental stages remain attached to the ovary through a cellular pedicel until completing stage V. Afterwards, they are liberated into the lumen and from there to the exterior. Some oocytes (classified as type VI) showed an atypical appearance indicating that some of the cellular components would be undergoing a degenerative process and/or reabsorption.     

Vitellogenesis in the tick Amblyomma triste (Koch, 1844) (Acari: Ixodidae) Role for pedicel cells

This study presents new information on the vitellogenesis of the tick Amblyomma triste. In this species, the ovary consists of a layer of epithelial cells, which form the ovarian wall, oogonia and developing oocytes; and the pedicel, a cellular structure that synthesizes and provides yolk precursors for developing oocytes. The pedicel also attaches oocytes to the external surface of the epithelial wall. In this study, evidence is provided in support of pedicel cells in providing compounds for oocytes during vitellogenesis.     

An ultrastructural characterization of the ooplasm in ovarian follicles of the immature ostrich (Struthio camelus)

Primordial, pre-vitellogenic and vitellogenic follicles were present in the ovary of the immature ostrich. The oocytes of these follicles were composed of a nucleus surrounded by ooplasm. Central, intermediate and cortical regions formed the ooplasm. The organelles present in these ooplasmic regions varied depending on the stage of follicular development. In primordial and small pre-vitellogenic (100-150 microm in diameter) follicles the central region of the ooplasm was dominated by an accumulation of organelles, which formed Balbiani's vitelline body. In contrast, the central region in vitellogenic follicles was filled with numerous large yolk spheres, many of which contained lining bodies. Numerous lipid droplets interspersed with mitochondria and small yolk spheres formed the intermediate ooplasmic region in primordial and small pre-vitellogenic follicles. In large pre-vitellogenic (150-400 microm in diameter) and vitellogenic follicles the intermediate region contained a greater density of mitochondria and small yolk spheres. Small yolk spheres were observed in the cortical region of pre-vitellogenic follicles. An interesting feature of the cortical region in vitellogenic follicles was the frequent occurrence of Golgi complexes. The results of the study indicate that although the ovarian follicles in the immature ostrich are not ovulated, the components and composition of the ooplasm are similar to those observed in the mature follicles of other avian species.     

Oogenesis is accompanied by cyclic morphological changes in hepatocytes of Neotropical freshwater fish Piabina argentea

We determined for the first time the reproductive biology of Piabina argentea through macroscopic and microscopic analysis of ovaries and evaluated the morphological changes in hepatocytes. Two hundred and 46 specimens were collected, 204 females and 42 males, between March 2014 and February 2015. Biometrics data were obtained. From females, gonad and liver samples were conducted to histological, histochemical and immunohistochemical techniques. Mature ovaries were used to determine absolute and relative fecundity. Total length and body weight values indicated that females were larger than males. The estimated weight–length ratio showed negative allometric growth. The absolute fecundity average was 171.83 ± 59.89 oocytes per ovary. In addition, females spawning capable and regressing stages were found throughout the sampling period and the presence of all oocyte types in regressing stage ovaries indicated asynchronous oocyte development and multiple spawning. From regenerating to spawning capable stage the oocytes accumulated yolk in cytoplasm became bigger. While in the liver hepatocytes with a larger cell area during regenerating stage and proliferative activity in the spawning capable stage were observed. Thus, our results indicate that P. argentea had an opportunistic reproductive strategy and cyclic morphological changes of hepatocytes occurred during the oogenesis.     

Risk factors to tick infestations and their occurrence on horses in the state of São Paulo, Brazil

From December 1998 to March 1999, 40 stud farms were studied in the state of S?o Paulo, Brazil. During visits to farms, horses reared under grazing conditions were examined for the presence of ticks. On each farm visit, horse pastures were closely inspected and a questionnaire was given to the farm supervisor with the purpose of gaining information about ecological and management variables (independent variables) that could be associated with the presence and infestation levels of ticks on the farm (dependent variables). Three tick species were found during the study. Anocentor nitens, Amblyomma cajennense and Boophilus microplus were present on horses from 38 (95%), 20 (50%) and four (10%) farms, respectively. All farms that had A. cajennense or B. microplus infestations also had A. nitens infestations. Only one of the four farms with B. microplus infestations on the horses also had A. cajennense infestations. Two farms had all horses free of ticks. There was a strong association between the presence of infestation by B. microplus on horses and the simultaneous use of a grazing area by cattle and horses (P = 0.000). There was no statistical association between any of the independent variables and the presence or infestation level of A. nitens on the horses (P > 0.20). The presence of A. cajennense was statistically associated with the presence of at least one mixed overgrowth pasture in the farm (P = 0.001). A mixed overgrowth pasture means the presence of undesired plants such as bushes and shrubs in the pasture. The presence of high levels of A. cajennense on horses was also associated with the presence of at least one mixed overgrowth pasture in the farm (P = 0.026). The regular use of acaricides was statistically associated with the presence of ticks on the horses (P < 0.05), making this procedure a result of the inefficacy of controlling ticks on the farms. The occurrence of human infestation by ticks was statistically associated with the presence of A. cajennense on the horses (P=0.000). The presence of at least one mixed overgrowth pasture on the farm was associated (P = 0.000) to either higher horse densities and to farms that did not mow all the pastures once a year, indicating that mowing all the pastures at least once a year can be considered a protective factor against the presence of mixed overgrowth pastures on the farm, and consequently, against the presence of A. cajennense on the horses.     

Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach

In this study, the fluorescent lipid dye Nile Red, was used to demonstrate that the lipid content of immature bovine oocytes is correlated with the morphological appearance of the ooplasm. Oocytes with a uniform dark cytoplasm contained significantly more intracellular lipids in lipid droplets compared with oocytes with a granulated or pale cytoplasm (p < 0.05). Furthermore, this lipid-analysing technique was applied for the first time on single bovine in vitro embryos, showing a significant increase of the lipid content in lipid droplets after culture in the presence of serum (p < 0.05).     

家蚕突变矮小卵血液蛋白及卵黄蛋白的研究

对家蚕突变矮小卵 (emi)品种在卵母细胞形成时期的血液、卵巢及卵中的总蛋白进行SDS PAGE发现 :在emi的卵巢及卵中含有卵黄蛋白 (Vn)、卵特异性蛋白 (ESP)和大部分的低分子量脂蛋白 (LP) ;emi蚕在蛹期和蛾期的血液中有卵黄原蛋白 (Vg)和LP ,蛹后期大部分emi血液中的Vg和LP含量逐渐减少 ,化蛾时血液中仅含少量的Vg和LP ,而少数emi个体的卵母细胞大量退化 ,在蛹后期和蛾期血液中还含有大量的LP和较多的Vg ;emi蚕从吐丝当天到蛾期的血液中的LP中有分子量为 30kD的蛋白带 ,在卵中却没有。以上结果表明 ,emi基因可能不仅决定卵大小 ,还可能对LP的不同成分的吸收 (如不能吸收LP中分子量为 30kD的蛋白 )有关 ,推测这可能是导致emi个体大部分不能正常发育的原因之一     

Interactions between the oocyte and surrounding somatic cells in follicular development: lessons from in vitro culture

Mammalian oogenesis occurs concomitantly with folliculogenesis in a coordinated manner in the ovaries. In vitro growth (IVG) culture systems of the oocytes have been developed as a new technology for utilizing incompetent oocytes in the ovary as a source of mature oocytes as well as for studying oogenesis, folliculogenesis, and oocyte-somatic cell interactions. The results of IVG experiments have suggested that direct association of oocytes and surrounding granulosa cells supports oocyte viability and growth through the gap junctions, which are efficient conduits for low molecular weight substances. It has been revealed that granulosa cells metabolize some molecules which are in turn transported into the oocytes. IVG systems have also provided evidence that FSH promotes the development of follicles at secondary or later stages by its stimulation of proliferation and differentiation of granulosa cells, and perhaps by its anti-apoptotic effects. In addition, interactions between granulosa cell-derived KIT ligands and oocyte KIT receptors have been suggested as initiating oocyte growth and follicular development. Furthermore, recent findings suggest there are growth factors derived from oocytes such as GDF-9 and BMP-15. With such factors, oocytes participate in follicular development by regulating the differentiation of surrounding somatic cells. These bidirectional communications between oocytes and somatic cells are important for oocyte growth and follicular development. IVG systems should provide further information regarding oogenesis and folliculogenesis in the ovary.     

Localization of thrombospondin‐1 and its receptor CD36 in the ovary of the ostrich (Struthio camelus)

Angiogenesis, the formation of new blood vessels from pre‐existing vasculature, plays a decisive role for the rapid growth of avian follicles. Compared to mammals, few data on the angiogenesis in the avian ovary are available. However, whereas several pro‐angiogenic factors in the avian ovary have been recently studied in detail, little information is available on the localization of anti‐angiogenic factors. The aim of this study was to determine the localization and possible function of the anti‐angiogenic factor thrombospondin‐1 (TSP‐1) and its receptor CD36 in the ovary of the ostrich using immunohistochemistry and to correlate the results with ultrastructural data. Whereas the oocytes and granulosa cells of all follicular stages were negative for TSP‐1, myofibroblasts of the theca externa and smooth muscle cells of blood vessels showed distinct reactions. A distinctly different staining pattern was observed for CD36. The oocytes were CD36 negative. No immunostaining for CD36 could be observed neither in the granulosa cells nor in the adjacent theca interna of vitellogenic follicles. In the theca externa, blood vessels protruding towards the oocyte showed CD36‐positive endothelial cells. In conclusion, a fine balance between angiogenic and anti‐angiogenic processes assures that a dense net of blood vessels develops during the rapid growth of a selected follicle. Anti‐angiogenic molecules, such as TSP‐1 and its receptor CD36 may, after the oocyte has reached its final size, inhibit further angiogenesis and limit the transport of yolk material to the mature oocyte. By this mechanism, the growth of the megalecithal oocyte during folliculogenesis may cease.     

Cross-reactivity between antigens from Amblyomma cajennense and A. hebraeum (Acari: Ixodidae)

Laboratory animals exposed to feeding ticks develop resistance which is reflected by a decline in tick engorgement weight, egg-laying by adults and reduced egg viability. Serum antibodies from these hosts and their reaction with tick antigens have been detected by different methods, including precipitation techniques, immunofluorescent techniques, ELISA and Western blots. However, little is known about the effects of antibodies on ticks that engorge on resistant hosts, or which tissues of the tick body are possibly immunogenic. Some researchers, using immunohistochemistry, have detected host antibodies in the gut, salivary glands and haemolymph of ticks engorged on resistant animals. The same technique has helped considerably in determining antigenic sites or antibody targets in other arthropods. Consequently, immunohistochemistry techniques were used in this study to detect cross-reactivity between sera raised against Amblyomma cajennense (Fabricius, 1787) with Amblyomma hebraeum (Koch, 1844), and vice versa. The results show the existence of shared antigens between the 2 tick species. In general, our results point more to a 1-way cross-reactivity of A. hebraeum with A. cajennense than a reciprocal cross-reactivity, suggesting that A. hebraeum is more immunogenic than A. cajennense.     

Prediction of the oocyte recovery rate in the bitch

In order to investigate the possibility of predicting the recovery rate of oocytes for use in a sperm-zona pellucida binding assay, ovaries were obtained from 67 bitches of 37 different breeds, and cumulus-oocyte complexes (COCs) were recovered by mincing the ovaries with a scalpel. The mean number of COCs recovered was 37.2 +/- 34.1 (range 0-145) per ovary. Age significantly affected COC recovery rates. From bitches 1-6 years old, 54.2 +/- 35.1 COCs/ovary were recovered, compared to 26.4 +/- 29.0 from bitches 7-13 years old (P = 0.003). The morphology of the uterus or the presence or absence of ovarian structures had no significant effect on COC recovery rates, although there was a tendency for more COCs to be recovered from ovaries with only follicles visible on the surface. There were no significant correlations between body weight or ovarian weight and COC recovery rates. There was a high correlation in the COC recovery rate between the two ovaries of a bitch, enabling an approximate estimation of the COC recovery rate from the second ovary when the COCs from the first ovary have been recovered. The large variation in COC recovery rates between bitches stresses the need for storage of canine oocytes in order to secure a high enough number of oocytes for a homologous sperm-zona pellucida binding assay in the dog.     

Expression of the orexigenic peptide ghrelin in the sheep ovary

Ghrelin has been implicated in the control of cell proliferation in reproductive tissue. Here, we provide evidence that both ghrelin mRNA and protein are present in ovarian follicles. Persistent expression of ghrelin was also demonstrated in sheep ovary throughout the estrous cycle and pregnancy. In fact, the relative mRNA levels of ghrelin varied depending on the stage of the cycle, with the highest expression during the development of the corpora lutea (CL) and minimal expression in the regressing CL. A similar pattern was seen during pregnancy. Dynamic changes in the profile of ghrelin expression during the estrous cycle and throughout pregnancy suggest a precise regulation of ovarian expression of ghrelin, which could represent a potential role for ghrelin in the regulation of luteal development. In conclusion, the presence of the ghrelin signaling system within the sheep ovary especially in the oocytes opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.     

Transferring and distributing profiles of p,p'-(DDT) in egg-forming tissues and eggs of laying hens following a single oral administration

Laying hens were administered orally with a single dose of p,p'-(DDT) (1 mg/kg bodyweight). The concentrations (microg/g) of DDT or its metabolites, p,p'-(DDE) and p,p'-(DDD), in the main tissues involved in egg formation (blood, liver, ovary, and oviducts) and egg yolk, collected 1 day after DDT dosing, were determined by normal-phase high-performance liquid chromatography. The limits of detection were 0.04 microg/g for DDT, 0.07 microg/g for DDE and 0.06 microg/g for DDD. In extractable fats from the above tissues and egg yolk, DDT and DDE were transferred/distributed throughout the tissues and egg yolk. DDD was detected only in the liver. The findings indicate that DDT is metabolized instantaneously to DDE/ DDD in the hen's body and they are transferred rapidly into the egg-forming tissues and egg yolk. Among the four tissues and yolk fats examined, the DDT levels were high in the ovary, oviduct and egg yolk; the DDE levels were high in the liver, ovary and oviduct and lowest in the yolk (P < 0.01).     

Evidence for a functional bone morphogenetic protein (BMP) system in the porcine ovary

Bone morphogenetic proteins (BMPs) play important roles in controlling fertility and ovulation rate. There is however, little information on the BMP system in the ovary of a large polyovular species. The aims of the present study were to investigate BMP-2 and -6 protein expression in the porcine ovary, their effects on granulosa cells in culture and their mechanism of action. Cells and oocytes were recovered from healthy antral follicles 2–6 mm in diameter. When assessed by Western blotting, oocytes and follicular fluid contained BMP-2 and -6. In addition, BMP-2 and -6 were observed in granulosa cells and BMP-2 was also found in theca cells. Granulosa cells were cultured in a serum-free system for 144 h in the presence of increasing doses (0, 3, 30 and 100 ng/ml) of BMP-2 or BMP-6. Both BMPs suppressed progesterone production in a dose-dependent manner after 48 h (P < 0.001) and 144 h (P < 0.05). Only BMP-6 stimulated cell proliferation at 100 ng/ml (P < 0.05). Investigation into the mechanism of action found that BMP-2 and -6 decreased cyclic adenosine monophosphate (cAMP) production (P < 0.01), expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) protein (P < 0.001) and steroidogenic acute regulatory protein (StAR) (BMP-6 only; P < 0.05). This supports the hypothesis that BMP-2 and -6 act as luteinization inhibitors. In conclusion, these findings provide evidence for the presence of a complex signalling mechanism in the porcine ovary and suggest that both BMP-2 and -6 may act in a paracrine manner to control granulosa cell function in this large polyovulatory species.     

In vitro maturation of oocytes derived from the brown bear (Ursus arctos)

This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete "rescue" techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 x 1.8 cm (range: 2.0-3.3 x 1.5-2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO2 for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 microg/ml estradiol-17beta, and 10 microg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 microsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 +/- 6.0 and 135.3 +/- 7.5 microm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 +/- 4.9 and 118.7 +/- 7.5 microm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages.     

Expression of the Orexigenic Peptide Ghrelin and the Type 1a Growth Hormone Secretagogue Receptor in Sheep Oocytes and Pre-implantation Embryos Produced In Vitro

Recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we provide evidence that both ghrelin and its receptor GHSR-1a (the type 1a growth hormone secretagogue receptor) are expressed at both mRNA and protein levels in sheep oocytes and pre-implantation embryos produced in vitro . Real-time RT-PCR experiments confirmed that ghrelin mRNA levels varied depending on developmental stage, with the highest expression in metaphase II (MII) oocytes, higher expression at the 2-cell stage, and minimal expression in germinal vesicle (GV) oocytes, 4- and 8-cell stages, and in the blastocyst. The levels of GHSR-1a mRNA decreased from GV to MII, increased immediately at the 2-cell stage and then remained stable until the blastocyst stage. Ghrelin protein was detected mainly in the cytoplasm close to the plasma membrane in both inner cell mass and trophectoderm cells, while GHSR-1a protein was most abundant in the plasma membrane. In conclusion, the presence of the ghrelin signalling system within the sheep oocytes and pre-implantation embryos opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.     

屠宰牛卵母细胞体外成熟和体外受精研究

对采集到的卵巢采取抽吸法、割破法回收卵母细胞,割破法得到的A级卵母细胞显著高于抽吸法。把有黄体和无黄体的卵巢卵母细胞进行培养比较,无黄体的卵巢卵母细胞的成熟率为84．33％,显著高于有黄体卵巢卵母细胞的74．32％。本试验中卵泡卵母细胞和深层卵母细胞体外成熟率均能达到75％以上．卵巢表面卵泡卵母细胞的卵裂率为54．76％,高于深层卵母细胞的卵裂率,但二者无显著差异。IVM时添加卵丘细胞、IVC时添加输卵管上皮细胞是比较理想的培养系统。     

RT-PCR技术检测绵羊卵巢内Ghrelin的表达

为了明确Ghrelin在绵羊卵巢有腔卵泡上是否存在表达,本研究利用实时定量RT-PCR技术检测了绵羊卵巢有腔卵泡内的卵母细胞、卵丘细胞和壁层颗粒细胞的Ghrelin蛋白的表达量情况。结果揭示绵羊卵巢有腔卵泡内各类型细胞Ghrelin mRNA的相对表达量大致相同。绵羊卵巢有腔卵泡内各类型细胞Ghrelin蛋白的表达及Ghrelin mRNA的表达模式,尤其是卵母细胞中的表达,揭示这一新型分子在绵羊卵巢上具有潜在的调控作用。     

哺乳动物腔前卵泡的分离与培养

哺乳动物卵巢中绝大多数卵母细胞以无腔形式存在，有腔卵泡所占比例很少。通过建立腔前卵泡的培养体系，获取大量的具有成熟和受精能力的卵母细胞，将极大地促进体外受精、核移植等胚胎工程技术的发展，并有利于研究卵泡和卵母细胞的发育规律。