Cellular responses of the skin and changes in plasma cortisol levels of trout (Oncorhynchus mykiss) exposed to acidified water

The skin structure and the plasma cortisol levels of trout, Oncorhynchus mykiss, were examined during 7 days of exposure to water of pH 5. By day-4 and-7, the thickness of the epidermis was significantly (P<0.05) less in acid exposed fish than in controls, and degenerative cells were common in the upper epidermal layers. Many epidermal cells exhibited signs of necrosis, and by day-7 many apoptotic cells were also present. Secretory vesicles of high electron density were abundant in the filament cells of the 3–4 outermost layers of epidermis, and intercellular spaces had increased. Mitotic figures occureed throughout the epidermis, with the exception of the outermost cell layer. Mucous cells became elongated after day-1, and later, newly differentiating mucous cells could be seen close to the skin surface, and many mucocytes contained mucosomes of high electron density. Rodlet cells were occasionally seen. Chloride cells appeared similar to those of control fish. Many leucocytes, mainly macrophages and lymphocytes, had penetrated the epidermis via the highly undulating basal lamina, and at day-7, numerous apoptotic lymphocytes were found. In the dermis, melanosomes became dispersed in the cytoplasmic extensions of melanocytes which were present in the epidermis of all acid-exposed fish. Iridocytes were rate after day-4, while fibroblasts were abundant and secreted large amounts of collagen. After 1 day of exposure to acidified water, a significant (P<0.05) elevation of the plasma cortisol level had occurred, but this subsequently declined, and had returned to control values by day-7. The changes in skin structure, however, remained throughout the whole exposure period.     

Cellular responses in the skin of the trout (Oncorhynchus mykiss) exposed to temperature elevation

The skin of rainbow trout was examined at the ultrastructural and cytochemical level after a 3–h exposure to an elevation of the water temperature, from 15 to 22° C. Within 3 h, the thickness of the epidermis had significantly ( P <0·05) decreased when compared to control fish. After 24 h it was restored, and from day 4 onwards even increased above control levels. The thickening of the epidermis was associated with appearance of many mitotic cells, not observed in control fish. Within 24 h many apoptotic epidermal cells were found, indicating enhanced ageing of the cells. Filament cells from the outer epidermal layers synthesized vesicles with peroxidasc activity within 3 h after temperature elevation. This enzyme was found also in apoptotic as well as in necrotic filament cells. Mucous cells became elongated and their mucosomes displayed peroxidase activity. Occasionally electrondense, probably serous, mucosomes appeared. In the epidermis rodlet cells were found. Both epideimis and dermis, became invaded by many lymphocytes and macrophages. The latter contained vesicles with peroxidase activity. Pigment–containing cytoplasmic extensions of melanocytes penetrated the epidermis while iridocytes disappeared from the dermis. The synthetic activity of dermal fibroblasts was stimulated. These results show that a moderate temperature elevation has pronounced and prolonged effects on the skin of the exposed fish. The effects are to a high extent comparable with those of stressors such as heavy metals, acid water or wounding.     

Waste-water management plant effluents cause cellular alterations in the skin of brown trout

To assess the impact of a sewage plant on fish, brown trout Salmo trutta were kept in two cages for 55 days in a moderately polluted river upstream of a sewage plant. In one of the cages, undiluted treated waste water of the sewage plant (WWE) was added at an average concentration of 5%, whereas the other cage received river water (R) only. A high mortality occurred in the WWE group. In comparison to control trout held in tap water, the skin structure and ultrastructure were altered clearly in both groups exposed to river water, including necrosis, apoptosis, decreased number of mucous cells, decrease in epidermal thickness, invasion of leucocytes, extension of melanocytes into the epidermis, being gradually more prominent in the WWE group. The most obvious difference between the two exposed groups was found in structure, size and electron density of the secretory vesicles of the filament cells. This and the observed vacuolation of Golgi saccules are indicative for disturbances in the secretory pathway of the filament cells. Certain toxins were suspected to cause the decompaction of myelin sheaths demonstrated in both groups. Reasons for the rather minor overall differences between the exposed groups are discussed. The extremely high mortality rate in the WWE group supports the importance of reducing the load of pollutants in the effluent of the waste-water management plant.     

Collagen degradation in the rabbit skin during short-term tissue culture

Full thickness rabbit skin explants were cultured on plastic dish for 1 week and the sequential morphological changes were examined daily by light and electron microscopy. During the cultured period, bundles of dermal collagen fibres gradually loosened and were removed from the upper dermis and from the cut margin of the explant, which was covered by a sheet of migrating epidermal cells. In these areas, cells containing phagocytosed collagen fibrils were observed from the 3rd day to the end of the culture period. These cells containing phagocytosed collagen fibrils included dermal fibroblasts and macrophages, epidermal keratinocytes and endothelial cells lining blood vessels. The presence of acid phosphatase activity in vacuoles containing the collagen fibrils suggested that intracellular degradation of collagen was occurring. In addition, extracellular collagen degradation was recognized around fibroblasts and beneath the migrating epidermis by the high collagenolytic activity at these sites. These findings suggest that both intra- and extracellular collagen degradation may participate in collagen removal from dermal connective tissue in cultured skin explants.     

20-羟基蜕皮甾酮处理后舞毒蛾外部形态和幼虫体壁超微结构的变化

为了探明20-羟基蜕皮甾酮对昆虫蜕皮过程中体壁的表皮层、 皮细胞及其细胞器的具体影响过程, 本研究利用透射电镜技术研究了20-羟基蜕皮甾酮对舞毒蛾Lymantria dispar （Linnaeus）5龄幼虫体壁超微结构的变化。结果表明, 用高浓度20-羟基蜕皮甾酮溶液浸过的白桦叶片饲喂幼虫, 处理6 h, 摄入约400 μg 20-羟基蜕皮甾酮后, 幼虫停止取食; 处理12 h时表皮细胞顶膜上的微绒毛减少, 在皮细胞与旧表皮之间形成蜕皮间隙, 旧头壳从幼虫头部脱离; 处理24 h时蜕皮间隙继续增大, 旧表皮与皮细胞进一步分离, 新表皮质层开始形成; 处理36 h时皮细胞顶膜形成较短的微绒毛, 胞质区域出现数量较多的电子疏松泡, 新表皮由上表皮、 外表皮及8层左右内表皮片层组成; 处理48 h时顶膜与内表皮界限模糊, 内表皮继续合成至16层左右; 72 h时细胞内出现大面积电子疏松泡, 内表皮合成至20层左右。 处理96 h时, 与对照组相比, 皮细胞细胞器较少, 核仁周围出现小部分空白区域, 胞质区域内含物减少; 虫体发黑缩小, 即将死亡; 内表皮层数仍旧保持20层左右。对照组幼虫6-96 h虫体活跃, 正常取食, 外部观察及透射电镜结果均未显现蜕皮现象; 表皮层由上表皮、 外表皮及内表皮组成; 皮细胞顶膜微绒毛密度高; 表皮细胞分泌活动旺盛, 胞质区域细胞界限明显, 内含物丰富; 细胞器典型而且活跃; 内表皮片层随时间不断增加至50层左右。结果提示, 外源20-羟基蜕皮甾酮能够导致舞毒蛾5龄幼虫的致死性蜕皮。     

Comparative study of Langerhans cells in normal and pathological human scars. I. Atrophic scars.

In the present study, we investigated Langerhans cells (LCs) in the epidermal component of human atrophic scars, comparing them with those in control skin and normotrophic scars. A preliminary analysis of the histological features was first carried out on vertical serial sections, stained with hematoxylin and eosin. The total epidermal thickness and the thickness of the single epidermal layers were then measured, by means of a digitizing tablet and a morphometric program run on an Apple IIe computer. These parameters were found to be significantly lower (40%) in atrophic scars, if compared to control skin and normotrophic scars (p less than 0.05). CDla-positive and HLA-DR-positive LCs were marked by indirect immunofluorescence. Their position among the epidermal layers, their dimensions, their density and their morphology were examined. In atrophic scars, LCs were densely and evenly distributed in all the epidermal layers. Their density was increased (about 1200 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both 300-400 cells/mm2 of epidermal area; p less than 0.001). The CDla-positive definite cell bodies, exhibiting an unstained nucleus, were as large as those evidentiated in the normotrophic scars and twice as much the control skin values (p less than 0.001). The present results provide morphological data that distinguish atrophic scars from control skin and normotrophic scars, and suggest an involvement of the Langerhans cells in this particular case of pathological scarring.     

Comparative study of Langerhans cells in normal and pathological human scars. II. Hypertrophic scars.

Langerhans cells (LCs) seem to play a crucial role in the immune system of the skin. Changes in their density, distribution, phenotype and/or morphology have been described in a number of skin diseases, mostly immunologically mediated. For this reason, we investigated LCs in human hypertrophic scars, since these scars are presently believed to have an immunological basis. A preliminary analysis of the histological features was carried out on vertical serial sections, stained with hematoxylin and eosin. Both epidermal and dermal components of hypertrophic scar biopsies were examined. The total epidermal thickness and the thickness of the single epidermal layers were also measured; the values obtained were similar to those of control skin and normotrophic scars. Subsequently, CDla-positive LCs, revealed by indirect immunofluorescence and immunoperoxidase techniques, were studied to determine their position among the epidermal layers and within the dermis, their dimensions, their density and their morphology. According to these observations, two main types of hypertrophic scars were identified. In the first type (7 scars), LCs were widely clustered within both the whole epidermis and the dermis. Their density was increased (about 750 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both about 400 cells/mm2 of epidermal area; p less than 0.001). The epidermal cell profiles, nearly three times larger than those of control skin, exhibited a dense network of interconnected dendrites. Further analysis for the presence of HLA-DR molecules revealed an anomalous expression of these antigens on keratinocytes. In the second type (3 scars), LCs density within the stratum Malpighii was unchanged, relative to control skin and normal scars, while CDla-positive cell bodies remained numerous in basal position and within the subpapillary corion. Epidermal LCs, only slightly larger than those evidentiated in control skin, displayed short and retracted dendritic projections. The aberrant expression of HLA-DR antigens on keratinocytes was very weak and sparse. The present results strongly suggest an immunologically activated state of the tissues examined; they provide morphological data that support the involvement of the immune system in hypertrophic scarring.     

Response of club cells in the skin of the carp Cyprinus carpio to exogenous stressors

The ultrastructure of club cells and neighbouring filament cells and leucocytes in the epidermis of carp, was studied under normal conditions and after exposure to several stressors: acid water, heavy metals, organic manure, brackish water and wounding. The effects of the stressors were remarkably similar. The club cells increased in size and contained more endoplasmic reticulum and Golgi areas. In both control and stressed fish, most mitotic figures of the filament cells were found adjacent to club cells, as was demonstrated after colchicine injection. Whereas in the controls apoptosis of filament cells was scarce and limited to the upper layer of the epithelium, in the stressed fish it was commonly seen in close proximity to the club cells but not in other mid-epidermal parts of the epithelium. This indicates that club cells influence the cellular kinetics of the filament cells. Under stress conditions leucocytes infiltrated the epidermis. Some were seen inside club cells. Apparently these leucocytes were taken up in phagosomes and subsequently they showed signs of necrotic degeneration. Leucocyte incorporation and degeneration in club cells were not observed in control fish. Control of the cellular turnover of filament cells and the elimination of leucocytes may represent new functions for club cells, which have mainly been associated with the production of pheromones.     

Stratification, specialization, and proliferation of primary keratinocyte cultures. Evidence of a functioning in vitro epidermal cell system

A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.     

新疆12种黄芩属植物叶表皮微形态结构的研究

应用光学显微镜和扫描电镜技术对新疆12种黄芩属植物叶片上的微形态特征进行观察。结果表明:该属植物叶的上表皮细胞形状及垂周壁式样有多种形式;而下表皮细胞形状均为不规则形,垂周壁式样均为深波形,不具分类学意义,但叶片两面分布的气孔器,在不同种间气孔大小、气孔密度、气孔指数、气孔外拱盖内缘等方面都存在着显著差异;其表皮角质层纹饰和表皮毛的微形态也各有不同;大多数植物叶片表面具腺点,其大小、分布及疏密程度也有不同。植物叶表皮上的这些微形态特征,可为探讨本属种间的分类学及亲缘关系提供一定的佐证。     

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.This work was partially funded by the European Commission (QLRT-2001-00722). A. Cuesta and I. Salinas are fellows of Fundación CajaMurcia and Fundación Séneca, respectively.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

Collagen fibril diameters increase and fibril densities decrease in skin subjected to repetitive compressive and shear stresses

Understanding microstructural changes that occur in skin subjected to repetitive mechanical stress is crucial towards the development of therapies to enhance skin adaptation and load tolerance in patients at risk of skin breakdown (e.g. prosthesis users, wheelchair users). To determine if collagen fibril diameter, collagen fibril density, dermal thickness, epidermal thickness, basement membrane length, and dermal cell density changed in response to repetitive stress application, skin subjected to moderate cyclic compressive and shear stresses for 1 h/d, 5 d/week, for 4 week was compared with skin from an unstressed contralateral control. The lateral aspects of the hind limbs of 12 Landrace/Yorkshire pigs were used. Skin from under the stressed site and a contralateral control site was processed for electron microscopy and light microscopy analysis. Electron microscopy results demonstrated significant (p<0.01) increases in collagen fibril diameter of 15.9%, 22.4%, and 22.9% for the upper, mid, and lower layers of the dermis, respectively, for the stressed skin compared with the control skin. Collagen fibril density (fibrils/unit cross-sectional area) decreased significantly for stressed vs. control by 19.8%, 29.2%, and 31.8% for the upper, mid, and lower layers, respectively. Light microscopy results demonstrated trends of a decrease in dermal thickness and an increase in cell density for stressed vs. control samples, but the differences were not significant. Differences in epidermal thickness and basement membrane length were not significant. These results demonstrate that quantifiable changes occur in collagen fibril architecture but not in the gross tissue morphology following in vivo cyclic loading of pig skin.     

Cytochemical and electron microscopic research on the peripheral blood leukocytes and skin fibroblasts in mucopolysaccharidoses

Cytochemical studies of the peripheral blood leucocytes in patients suffering from mucopolysaccharidoses (MPS) have revealed metachromatic granules in the cell cytoplasm. Electron microscopy of these cells has shown multiple cytoplasmic vacuoles. It is supposed that metachromatic granules in blood leucocytes of patients with MPS observed in the photo-optical studies correspond to the vacuoles found under electron microscope. The obtained data have shown that peripheral blood leucocytes and skin fibroblasts have the common ultrastructure in MPS patients. The data of electron histochemical studies testify to that the vacuoles of skin fibroblasts are filled with glycosaminoglycans.     

Changes in gap junction distribution and connexin expression pattern during human fetal skin development.

Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.     

Ultrastructural demonstration of endogeneous peroxidase activity in mammalian epidermis

With the diaminobenzidine method, endogenous peroxidase activity was demonstrated in the nuclear envelope and in the endoplasmic reticulum of non-keratinized keratinocytes and Langerhans cells of the epidermis of the newborn mouse, adult guinea pig and man. In the guinea pig all non-keratinized layers of keratinocytes showed this enzyme activity, whereas in the two other species examined peroxidase activity was limited to the suprabasal layers. The most pronounced activity was found in the Langerhans cells. The melanocytes were negative. With the same method, cytochrome c/cytochrome oxidase activity could be localized in the mitochondria of all epidermal cells of mouse and man, but not in the guinea pig.     

Vertical distribution in and isolation of bacteria from Lake Vanda: an Antarctic lake

Vertical distribution of bacteria in Lake Vanda, an Antarctic meromictic lake, was examined by the acridine orange epifluorescence direct count method. Total bacteria were 10⁴–10⁵ cells · ml^–1 in the water at 55 m depth and above, and increased drastically to 10⁷ cells · ml^–1 in the bottom water. Filamentous or long rodshaped bacteria occurred at a high frequency in the upper layers, but in the bottom layers most bacteria were coccoidal or short rods. Mean bacterial cell volume in water of between 10 m and 60 m deep was fairly large compared with common bacterial populations in seawater and lake water. Aerobic heterotrophic bacteria were recovered from the water of a depth of 30 m and above, and were assumed to belong to Caulobacter. Viable heterotrophic bacteria were not recovered from the high salinity deep water by media prepared with the same deep water. Phototrophic purple non-sulphur bacteria were isolated by enrichment cultures from water at 55 m depth.     

Precocene-induced necrosis and haemocyte-mediated breakdown of corpora allata in nymphs of the locust Locusta migratoria

Light and electron microscopic studies, including the use of the vital dyes trypan blue and acridine orange, indicate that the topical application of precocene II rapidly triggers degenerative processes in the corpora allata (CA) leading within a few days to the virtual disappearance of the parenchymal cells of these glands. The following sequence of events was observed: 1) Within 2 h, many of the cells in fixed nymphal specimens showed increased electron density. During the next few hours, they decreased considerably in volume (shrinkage necrosis). Intercellular spaces increased simultaneously. Although a variable number of cells remained electron lucent, they showed nuclear and cytoplasmic degeneration later on (coagulative necrosis). 2) Haemocytes arrived at the CA sheath and invaded the CA in large numbers after 12 h. 3) CA cells became increasingly necrotic, and cell fragments were budded off and were phagocytosed most noticeably after 1 to 3 days. Thereafter no CA parenchyma remained. 4) Haemocytes dispersed.     

Identification of functional markers in a self-assembled skin substitute in vitro

Some functional parameters were identified and assessed in a tissue-engineered self-assembled skin substitute. This skin substitute was produced using fibroblasts and keratinocytes isolated from adult human skin. Keratinocytes were seeded on a dermal layer, composed of two fibroblast sheets cultured for 35 d. The epidermal cells formed a stratified and cornified epidermis and expressed differentiation markers, notably involucrin and transglutaminase. Interestingly and for the first time, the receptor for vitamin D3 was detected in all of the epidermal cell layers of the skin substitute, as it is reported for normal human skin. This observation suggests that keratinocytes retain key receptors during their differentiation in the skin model. A network of collagen fibers was observed by electron microscopy in the dermal layer of the model. In the dermis, collagen fibers remodeling and assembly is dependent on enzymes, notably prolyl-4-hydroxylase. For the first time in a skin construct, the expression of prolyl-4-hydroxylase was detected in dermal fibroblasts by in situ hybridization. The secretion of collagenases by the cells seeded in our skin substitute was confirmed by zymography. We conclude that the self-assembly approach allows the maintenance of several functional activities of human skin cells in a skin model in vitro.     

Tolerance in early embryo aggregation-derived mouse chimaeras.

Epidermal cell suspensions of 90% average viability prepared from adult mouse tail skin by trypsinization and glass wool filtration were compared with leucocytes for their capacity to induce and reveal cell-mediated cytotoxicity to histocompatibility antigens in an H-2 different strain combination. As targets in short-term chromium release assays, epidermal cells incorporated ten times more ⁵¹Cr than normal lymph node cells yet released a lower proportion spontaneously. Although they were more resistant to lysis than lymph node cells, they registered high levels of cytotoxicity when particularly active attacker cells were used, even at low attacker-to-target cell ratios. In mixed cell cultures, irradiated epidermal cells were as effective as spleen cells in boosting immunity induced in vivo by skin allografts. Epidermal cells also were effective primary immunogens in vitro, but at higher responder-to-stimulator cell ratios than required for spleen cells. The specificity of epidermal cells as both targets and immunogens fully paralleled that of leucocytes from the same donors.