Vitellogenin transport and yolk formation in the quail ovary

Morphological and biochemical investigations were made on the yolk formation in ovaries of the quail Coturnix japonica. Morphologically, two ways of nutrient uptake were observed in follicles. In small oocytes of white follicles, vitellogenin (VTG) was taken up through fluid-phase endocytosis which was assisted by follicular lining bodies. The lining bodies were produced in follicle cells. They adhered to the lateral cell membrane, moved along the membrane in the direction of the enclosed oocyte and were posted to the tips of the microvilli. These tips, now with lining bodies, were pinched off from the main cell body, engulfed by indented cell membranes of the oocyte, and transported to yolk spheres. In large oocytes of yellow follicles, VTG and very-low-density lipoproteins (VLDL) were taken up through receptor-mediated endocytosis. The VTG and VLDL particles diffused through the huge interspaces between follicle cells, and once in oocytes were transported to yolk spheres via coated vesicles. Immunohistochemistry showed that the VTG resides on or near the surface of the follicle cell membrane at the zona radiata whereas the cathepsin D resides at or near the oocytic cell membranes. Tubular and round vesicles in the cortical cytoplasm of oocytes were also stained with both antisera, suggesting that these vesicles are the sites where the VTG is enzymatically processed by cathepsin D. Upon analysis by SDS-PAGE, a profile similar to that of yolk-granule proteins was produced by incubating VTG with a quail cathepsin D of 40 kD.     

Chicken oocyte growth: receptor-mediated yolk deposition

During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocyte's energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.     

Vitellogenin dynamics during egg-laying: daily variation, repeatability and relationship with egg size

Daily variation in circulating levels of the avian yolk precursor, vitellogenin (VTG), throughout the laying cycle was investigated in female zebra finches Taeniopygia guttata and compared with predicted ovarian follicle demand (based on a model of follicular development for this species). In general, the pattern of variation in plasma VTG matched the predicted demand from the developing ovarian follicle hierarchy. Plasma VTG was non-detectable in non-breeders, but increased rapidly with onset of yolk development, remaining high (1.43–1.82 μg/ml, zinc) through to the 3-egg stage. Plasma levels then declined at the 5-egg stage (to 0.78±0.32 μg/ml) and were undetectable at clutch completion. This result is consistent with the hypothesis that yolk precursor production is costly and that selection has matched supply and demand. While inter-individual variation in plasma VTG was marked (e.g. 0.47–4.26 μg/ml at the 1-egg stage), it also exhibited high intra-individual repeatability (r=0.87–0.93). Finally, we examined the relationship between plasma VTG and primary reproductive effort. While individual variation in plasma VTG was independent of clutch size, laying interval and laying rate, there was a complex, diet-dependent relationship between VTG and egg size, with low plasma VTG levels being associated with both very small (<0.90 g) and very large (>1.15 g) egg sizes.     

Experimental (tamoxifen-induced) manipulation of female reproduction in zebra finches (Taeniopygia guttata)

Experimental manipulation of reproductive phenotype is a potentially powerful approach for understanding the fitness relationships of traits such as egg size, egg composition, and egg number. In this study, I investigated the effect of the antiestrogen tamoxifen on multiple, estrogen-dependent reproductive traits in female zebra finches (Taeniopygia guttata). Short-term tamoxifen treatment of egg-laying females (two daily injections before laying) had no effect on the timing or the pattern of egg laying compared to sham controls. However, tamoxifen treatment caused (1) a marked, but transient, decrease in egg size; (2) increased within-clutch egg-size variation; (3) a reduction in plasma vitellogenin (VTG) levels; and (4) lower dry yolk and yolk protein content of tamoxifen-treated females. Plasma levels of the second yolk precursor, very low density lipoprotein (VLDL), were not affected by tamoxifen, and tamoxifen appeared to have no effect on oviduct function in egg-laying females. These results are consistent with tamoxifen blocking estrogen receptors in the liver, suppressing VTG production, and decreasing the plasma pool of yolk precursors below a level required to maintain yolk formation at the normal rate. Tamoxifen treatment can therefore be used successfully to manipulate several components of the female reproductive phenotype (egg composition, intraclutch egg-size variation) to further explore the fitness consequences of these traits.     

Corticosterone regulation of ovarian follicular development is dependent on the energy status of laying hens

Glucocorticoids participate in the arousal of stress responses and trigger physiological adjustments that shift energy away from reproduction toward survival. Ovarian follicular development in avians is accompanied by the supply of yolk precursors, which are mainly synthesized in the liver. Therefore, we hypothesized energy status and hepatic lipogenesis are involved in the induction of reproductive disorders by glucocorticoids in laying hens. The results show that corticosterone decreased the laying performance by suppressing follicular development in energy-deficit state, rather than in energy-sufficient state. In corticosterone-treated hens, the suppressed follicular development was associated with the reduced availability of yolk precursor, indicated by the plasma concentration of VLDL and vitellogenin and the decreased proportion of yolk-targeted VLDL (VLDLy). Corticosterone decreased the expression of apolipoprotein B and apolipoprotein VLDL-II in the liver. A drop in VLDL receptor content and an increase in the expression of tight junction proteins occludin and claudin1 were also observed in hierarchical follicles. The results suggest corticosterone-suppressed follicular development is energy dependent. The decreased apolipoprotein synthesis and VLDLy secretion by liver are responsible for the decreased availability of circulating yolk precursor, and the upregulation of occludin and claudin expression further prevents yolk deposition into oocytes.     

Pituitary control of ovarian activity in the lizard, Agama agama

The principal ovarian structures in Agama agama are non-yolky and yolky follicles, corpora lutea and atretic follicles. Hypophysectomy affects all stages of vitellogenesis and follicular atresia results. Atresia involves resorption and digestion of yolk by the granulosa cells, which eventually undergo a fatty degeneration. Immature follicles and corpora lutea are not affected by hypophysectomy.
Injections of 0.2 mg FSH (NIH-FSH-S3 ovine) on alternate days for 14 days not only maintain vitellogenesis in hypophysectomized animals, but also tend to overstimulate the small follicles to synthesize yolk. A similar dose of LH(NIH-LH-SII ovine) fails to maintain vitellogenesis in hypophysectomized animals, while a higher dose of 0.4 mg LH tends to overstimulate the small follicles. An unusual rupture of yolky follicles in FSH injected lizards is reported and a possible development of such follicles into corpora lutea is discussed.     

Migratory constraints on yolk precursors limit yolk androgen deposition and underlie a brood reduction strategy in rockhopper penguins

Hormonally mediated maternal effects link maternal phenotype and environmental conditions to offspring phenotype. The production of lipid-rich maternal yolk precursors may provide a mechanism by which lipophilic steroid hormones can be transported to developing yolks, thus predicting a positive correlation between yolk precursors in mothers and androgen levels in eggs. Using rockhopper penguins (Eudyptes chrysocome), which produce a two-egg clutch characterized by extreme egg-size dimorphism, reversed hatching asynchrony and brood-reduction, we examined correlations between circulating concentrations of the primary yolk-precursor vitellogenin (VTG) and levels of yolk androgens. Previous work in Eudyptes penguins has shown that egg-size dimorphism is the product of migratory constraints on yolk precursor production. We predicted that if yolk precursors are constrained, androgen transport to developing yolks would be similarly constrained. We reveal positive linear relationships between maternal VTG and androgens in small A-eggs but not larger B-eggs, which is consistent with a migratory constraint operating on the A-egg. Results suggest that intra-clutch variation in total yolk androgen levels depends on the production and uptake of yolk precursors. The brood reduction strategy common to Eudyptes might thus be best described as the result of a migratory constraint.     

Interindividual variation in yolk mass and the rate of growth of ovarian follicles in the zebra finch (Taeniopygia guttata)

The amount of resources invested in an individual egg yolk must be determined by its rate of growth and/or the duration of growth. We examined interindividual variation in the growth rate of yolks by injecting radiolabeled amino acid into breeding female zebra finches and measuring the activity associated with protein in the yolks of eggs laid subsequently. We predicted that (1) there would be a positive correlation between yolk mass and the rate of uptake of activity into the yolk; and (2) there would be a negative correlation between clutch size and the amount of activity taken up by each of the follicles due to competition between follicles for circulating yolk precursors. The rate of uptake of activity by the yolks was positively related to yolk mass (r2=0.24, 0.35 and 0.50 for the yolks of the third-, fourth- and fifth-laid eggs, respectively), suggesting that interindividual variation in yolk mass is due, at least in part, to variation in the rate of follicle growth. However, we found no evidence of a trade-off between yolk size and number. The uptake of activity was generally repeatable between breeding attempts (repeatability= 0.23-0.44), as was mean yolk mass (repeatability = 0.35), suggesting that these traits are characteristics of individual females.     

Plasma zinc as an index of vitellogenin production and reproductive status in the domestic fowl.

1. A technique is described for the accurate determination of circulating vitellogenin concentrations by measurement of plasma zinc concentrations before and after selective precipitation of lipoproteins in the domestic fowl. 2. Vitellogenin zinc concentrations exhibit a high degree of correlation with those of another major egg yolk precursor, very low density lipoprotein (VLDL) in immature female birds, during developing and maximum egg production in mature birds and following cessation of egg laying. 3. Mature female patterns of plasma vitellogenin and VLDL were induced by injection of oestradiol 17 beta (5 mg/kg body weight per day for 3 days) into adult cockerels. 4. It is suggested that measurement of plasma zinc provides a simple and accurate technique for the estimation of vitellogenin production and reproductive status in the domestic fowl and that this may be applied to other oviparous vertebrates.     

Effects of 2,2'4,4'5,5'-hexachlorobiphenyl (PCB 153) on plasma sex steroids and vitellogenin in rockfish (Sebastes schlegeli)

We examined the estrogenic effect of 2,2'4,4'5,5'-hexachlorobiphenyl (PCB 153) on the rockfish, Sebastes schlegeli. We measured levels of plasma estradiol-17beta (E(2)) and testosterone (T) by radioimmunoassay (RIA). Plasma concentrations of T and 17alpha-hydroxyprogesterone in female and male fish injected with PCB 153 using two dosages (0.16 mg/kg body weight. and 0.57 mg/kg) did not differ significantly between sexes or from sham-injected controls of the same sex. Plasma concentrations of E(2) in females injected with PCB 153 (both levels) increased at 12 and 24 h. Concentrations of plasma vitellogenin (VTG) in females increased 72 h after injection with PCB 153 and reached 0.38 and 1.25 mg/mL, respectively. No VTG was detected in males injected with the same dosage. These results suggest that PCB 153 may lead to the production VTG in female rockfish through a synergistic effect with E(2), resulting in indirect disruption of the aromatization process.     

Accumulation of yolk in a caecilian (Gegeneophis ramaswamii) oocyte: A light and transmission electron microscopic study

There is a paucity of information on the female reproductive biology of the caecilian amphibians when compared with the other vertebrate groups. Hence, the accumulation of nutrient reserves in the form of yolk and formation of yolk platelets were studied in Gegeneophis ramaswamii, adopting light microscopic histological and transmission electron microscopy analysis. Previtellogenic as well as vitellogenic follicles were observed in appropriate preparations. On the basis of the source and the routes of entry, we identified four types of yolk precursor materials, precursors 1 to 4. The earliest material appearing in the oocyte consists of abundant lipid vesicles during the previtellogenic phase, i.e., much before the follicular epithelium is fully established. This is a contribution from the oocyte mitochondria, which we identified as yolk precursor material 1, and it is autosynthetic. Once the follicle cell‐oocyte interface is fully established, there is an accumulation of the principal component of the heterosynthetic yolk by sequestration from the blood through the intercellular spaces between follicle cells in a pinocytic process. This we identified as yolk precursor material 2. There was also an indication of a lipidic yolk material synthesis in the follicle cells sequestered from maternal blood through the follicle cells in an endocytic process in which the macrovilli of follicle cells and the microvilli of the oocyte play a role. This we identified as yolk precursor material 3. Contribution to the yolk of peptidic, glycosidic, and/or lipidic material synthesized in the vitellogenic oocyte was also indicated. This we identified as yolk precursor material 4. The sequential development of intercellular associations and indications of synthesis/sequestration of the yolk have been traced. Thus, we report the mechanistic details of synthesis/sequestration of the yolk materials in a caecilian. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Characterization of ovarian follicular dynamics in dromedary camels (Camelus dromedarius)

Ovarian follicular dynamics was monitored by transrectal ultrasonography, for a period of 60 to 90 days, and its correlation with plasma estradiol-17β (E2) and progesterone (P4) were studied in seventeen, multiparous, non-lactating, 12 to 20-year-old dromedary camels. The average number of follicles recruited (12.77 ± 0.93) in each wave between animals varied (P < 0.001). The number of follicles recruited during different follicular waves was highly repeatable (0.95) within individual animals. The growth and mature phase periods of the dominant follicle (DF) were 6.10 ± 0.15 and 10.20 ± 0.47 days, respectively with a linear growth rate of 1.17 ± 0.02 mm/day between Day 0 and 10 of the follicular wave. There was an inverse relationship between the diameter of the largest DF and number of follicles (r = −0.95, P < 0.001). The DF development did not regularly alternate between the ovaries and the incidence of codominance was 45%. The mean maximum diameter of DF during its mature phase was 27.30 ± 0.78 mm and oversized follicle was 38.43 ± 1.41 mm. In 73.3% waves, the DF continued its growth for a period of 10.64 ± 1.53 days even after losing its dominance and developed into oversized follicle. The duration of the regression phase of DF and oversized follicle were 24.71 ± 3.79 and 18.50 ± 2.23 days. The mean duration of a complete follicular wave was 47.11 ± 2.94 days with an interwave interval (IWI) of 16.36 ± 0.37 days. The IWI within an individual was repeatable (0.88) and between the animals was variable (P < 0.001). Plasma E2 concentration profiles showed a wave like pattern. The peak plasma E2 concentrations were attained approximately 12 days after beginning of the growth phase, when the largest DF grew to a diameter of 18.7 mm. Plasma concentration of P4 was below 1.0 ng/mL in 85% of waves and above 1.0 ng/mL in 15% of the waves for a period of 3 to 6 days in the absence of spontaneous ovulation. It is concluded that ovarian follicular development and plasma E2 concentrations occurs in a wave like pattern in dromedary camels and the IWI and follicle numbers recruited per wave are variable between the animals and repeatable within an individual animal.     

Histological and histochemical studies of post-ovulatory and pre-ovultory atretic follicles in Mustelus canis

The atresia of post-ovulatory and pre-ovulatory follicles of the viviparous smooth dogfish, Mustelus canis, is compared for approximately the first fourth of an 11 month gestation. A thick collagenous sheath and numerous tubules in the theca identify the large, folded stage A post-ovulatory follicle. In stage B the tubules have been filled by cells to form “islands.” In stage C the entire structure is greatly diminished, adjacent islands tend to fuse, the collagenous sheath is virtually gone and the granulosa is degenerating. Preovulatory follicles from large, yolky oocytes pass through four stages beginning with yolk phagocytosis by granulosa cells of the villi (stage I), which are long and granular in stage II; villi fuse, theca cells increase greatly, fill with granules (stage III), encroach on the granulosa and disperse it into small groups of cells which finally disappear (stage IV) leaving a mass of thecal cells. A special type of pre-ovulatory follicle from small non-yolky oocyte atresia exhibits prominent thecal tubules and an unusual arrangement of granulosa cells. This follicle appearrs to enlarge during the summer, becoming multilobed; few granules are present. The distribution of lipid in frozen sections, stained by Oil red O, is described for all types of follicles. Schultz and Lewis and Lobban tests for steroids were made on frozen sections with corresponding results. Positive green tests indicating the presence of steroids or possible steroidogenesis were limited to: (1) one post-ovulatory follicle, in the islands; (2) four stage III and seven late stage IV pre-ovulatory yolky atretic follicles; (3) two special atretic follicles. The special atretic follicle appears to be a unique feature of M. canis and it is suggested tentatively that it may be related to viviparity.     

Salt concentration-dependency of vitellogenin processing by cathepsin D in Xenopus laevis

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.     

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.)

The aim of this study is to assess, by RT‐PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis). J. Cell. Physiol. 228: 547–555, 2013. © 2012 Wiley Periodicals, Inc.     

Effects of pituitary glycoprotein hormones and thyroid hormones on in-vitro vitellogenin incorporation into organ-cultured oocytes in the Japanese eel, Anguilla japonica

The objectives of the present study were to establish a long-term culture system for previtellogenic ovarian fragments of the Japanese eel (Anguilla japonica) and to identify the effects of salmon pituitary glycoprotein fraction (SPG), thyroxine (T4), and 3,5,3'-triiodothyronine (T3) on the uptake of vitellogenin (VTG) by cultured ovarian fragments evidenced by the appearance of yolk globules (YGs) within the oocytes. Yolk globules first appeared in the oocytes incubated in media containing only VTG (VTG-only group) after 9 days, whereas YGs began to accumulate in the oocytes of ovarian fragments cultured in media containing VTG+SPG (SPG group) following only 3 days of incubation. Furthermore, the occurrence of vitellogenic oocytes (%VO) and proportion of YGs within oocytes (%YG area) were significantly higher in follicles cultured in 30 ng/ml SPG throughout the culture period. No such stimulatory effects of T4 on VTG uptake were observed. Incubation of ovarian fragments with VTG and T3 (T3 group; 50 ng/ml) resulted in an increased %VO compared to follicles in the VTG group by day 9 of culture, and from day 10 onwards, both %VO and %YG area became significantly higher in follicles of the T3 group. Interestingly, SPG stimulated VTG incorporation and YG accumulation even in small oocytes (approximately 150 microm), whereas T3 showed these effects only in larger sized oocytes (> 180 microm). These results suggest that both SPG and T3 can accelerate VTG incorporation, but the mechanisms whereby this is achieved may differ between these hormone preparations.     

Differential effects of RU486 and indomethacin on follicle rupture during the ovulatory process in the rat

Ovulation (i.e., the release of mature oocytes from the ovary) requires spatially targeted follicle rupture at the apex. Both progesterone and prostaglandins play key roles in the ovulatory process. We have studied follicle rupture and ovulation in adult cycling rats treated with a progesterone receptor antagonist (RU486), an inhibitor of prostaglandin synthesis (indomethacin, IM), or both. All rats were treated with LHRH antagonist on the morning (0900 h) of proestrus to inhibit endogenous gonadotropins and with 10 microg of ovine LH (oLH) at 1700 h in proestrus to induce ovulation. Animals were treated from metestrus to proestrus with 2 mg/day of RU486 or vehicle (olive oil) and on the morning of proestrus (1200 h) with 1 mg of IM or vehicle (olive oil). Some rats treated with vehicle or RU486 were killed on the morning of proestrus to assess preovulatory follicle development. The remaining rats were killed on the morning of estrus to study follicle rupture and ovulation. In vehicle-treated rats, oLH induced ovulation in 98% of follicles. In IM-treated rats, spatial targeting of follicle rupture was disrupted. Most oocytes were released to the ovarian interstitium (50%) or to the periovarian space (39%), and a smaller percentage (11%) of oocytes remained trapped inside the luteinized follicle. RU486-treated rats showed, on the morning of estrus, unruptured luteinized follicles. Only occasionally (2.8%), the oocytes were released to the periovarian space. IM treatment induced follicle rupture in RU486-treated rats, and 25% of oocytes were released to the ovarian interstitium. However, the number of oocytes released to the periovarian space (i.e., ovulated) was not increased by IM treatment in rats lacking progesterone actions. Overall, these data indicate that RU486 and IM have opposite effects on follicle rupture and suggest that both progesterone and prostaglandins are necessary for the spatial targeting of follicle rupture at the apex.     

Oogenesis of the mayfly Habrophlebia eldae: Synthesis of vitelline and chorionic envelopes

The ultrastructure of developing ovarian follicles inside the panoistic ovarioles of Habrophlebia eldae were examined to observe the events occurring during egg maturation up to the full formation of the chorionic envelopes. The early vitellogenic follicles are coupled by gap junctions and are extensively interlocked with the oocyte plasma membrane via microvilli. With the onset of vitellogenesis, coated pits and coated vesicles are precursors to yolk deposition and are visible at the follicle cell-oocyte interface. Postvitellogenic development entails the deposition of the egg envelopes. The vitelline envelope arises from the coalescence of rectangular plaques whose precursors are visible in Golgi complexes as heterogeneous electron-opaque granules. A chorionic pattern of ridges on the egg surface characterizes the shell of H. eldae. The fully developed chorion shows three distinct regions with differently organized patterns. A fine layer of fibrous material (a secretion of the follicle cells, Ephemeroptera devoid of accessory glands) adheres to the egg chorion and is probably involved in attachment to the substrate.