Effect of 5-Hydroxytryptamine on Steroidogenesis and Oocyte Maturation in Pre-ovulatory Follicles of the Medaka Oryzias latipes

The effect of 5-hydroxytryptamine (5-HT) on steroidogenesis and oocyte maturation in pre-ovulatory follicles of the medaka Oryzias lalipes was examined using in vitro culture system. The earliest breakdown of the germinal vesicle of intrafollicular oocytes occurred about 17 hr after the beginning of incubation in the presence of 5-HT at concentration of 10 ng/ml or more. 5-HT induced oocyte maturation in a dose-dependent manner. Cyanoketone inhibited this stimulation. The concentration of 5-HT required to induce oocyte maturation corresponded to that required to enhance the production (secretion) of estradiol-17β and 17α,20β-dihydroxy-4-pregnen-3-one by pre-ovulatory follicle cells. At a concentration of 1 μg/ml, the follicle had to be exposed to 5-HT for at least 4 hr for oocyte maturation accompanied by ovulation to occur. These results indicate that 5-HT induces in vitro maturation of medaka oocytes by stimulating 17α,20β-dihydroxy-4-pregnen-3-one production by pre-ovulatory follicular cells.     

Oogenesis in Fundulus heteroclitus. VI. Establishment and verification of conditions for vitellogenin incorporation by oocytes in vitro

A procedure was developed for studying vitellogenin (VTG) incorporation by vitellogenic oocytes of Fundulus heteroclitus in vitro. Since homologous VTG can be obtained from this animal only with great difficulty, the use of [32P]VTG from Xenopus laevis was explored as an alternative. Vitellogenic as well as maturational-stage oocytes were found to sequester X. laevis [32P]VTG from the medium, and incorporation was found to be linear with time for at least up to 12 hr. Once incorporated into the oocyte, [32P]VTG did not appear to undergo turnover. The effect of different [32P]VTG concentrations on incorporation indicated that the uptake mechanism was saturable. Unlabeled F. heteroclitus VTG and X. laevis VTG were also found to compete effectively with X. laevis [32P]VTG, whereas bovine serum albumin did not. These results represent the first documentation of a successful culture system for receptor-mediated VTG incorporation by teleost oocytes.     

The Influence of Yolk Protein Proteolysis on Hydration in the Oocytes of Fundulus heteroclitus

Growth in oocytes of many marine teleosts can be attributed to a combination of yolk accumulation during the vitellogenic phase of development and water uptake during meiotic maturation. In the salt marsh fish, Fundulus heteroclitus , hydration associated with maturation gives rise to a greater than two-fold increase in oocyte volume. It has been proposed that a concurrent proteolysis of specific yolk proteins may be the mechanism driving this water uptake. To test this hypothesis, we used various in vitro culture techniques to block or significantly reduce oocyte hydration while allowing meiotic maturation to continue, then examined yolk proteins by SDS-polyacrylamide gel electrophoresis. We were able to dissociate yolk proteolysis from both hydration and nuclear maturation stimulated by a maturation-inducing steroid, 17α-hydroxy- 20β-dihydroprogesterone. It therefore appears that the proteolysis of specific yolk proteins observed in maturing oocytes of marine teleosts is an independent developmental event, and is not directly involved in the hydration mechanism.     

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.     

Steroidogenesis in the Ovarian Follicle of Medaka (Oryzias latipes, a daily spawner) during Oocyte Maturation

The metabolism of ¹⁴C-labeled steroid precursors by cell-free homogenates of medaka ( Oryzias latipes , a daily spawner) ovarian follicles at 12 different developmental stages was examined using thin layer chromatography (TLC). The radioactive metabolites produced were identified and tested for their ability to induce germinal vesicle breakdown (GVBD) in oocytes in an in vitro homologous bioassay. When homogenates of follicles isolated during oocyte maturation were incubated with ¹⁴C-labeled 17α-hydroxyprogesterone, 13 metabolites were detected in TLC. Among these metabolites, one metabolite exhibited very high maturation inducing activity by the in vitro bioassay. This metabolite was identified as 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) by its chromatographic mobility in TLC and recrystallization to constant specific activity. 17α,20β-DP production was high in follicles collected between 10 and 6 hr before spawning. A much less biologically active metabolite, 17α,20β-dihydroxy-5β-pregnane-3-one appeared in follicles immediately after the formation of 17α,20β-DP. A similar pattern of steroidogenesis was observed when the follicles were incubated with ¹⁴C-labeled pregnenolone and progesterone. The timely synthesis of 17α,20β-DP in medaka at the onset of oocyte maturation, together with the demonstration that this progestogen is the most potent inducer of oocyte maturation in vitro , provides further evidence that 17α,20β-DP is the naturally occurring maturation-inducing hormone in the medaka. The results also suggest that the conversion of 17α,20β-DP to its 5β-reduced metabolite may be an inactivation process.     

Brief report: Synthesis of ribonucleic acid in oocytes collected from squirrel monkeys and humans following chorionic gonadotropin administration

The time between administration of human chorionic gonadotropin (hCG) and follicle aspiration was analyzed for alterations in [³H]uridine incorporation [as an indicator of relative synthesis of ribonucleic acid (RNA)] of oocytes from squirrel monkeys and humans. There was a significant decline in both the uptake and incorporation of [³H]uridine in squirrel monkey oocytes by 36 hr following hCG administration to the animals, as compared with 16 hr after hCG. Similarly, RNA synthesis diminished in oocytes collected 35 hr after hCG in humans, as compared with 12 hr after hCG. This reduction in RNA synthesis of maturing oocytes is similar to that of other mammalian species. This provides evidence for an increased interval between hCG administration and follicle aspiration in order to recover mature oocytes for in vitro fertilization studies.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

Shift in Steroidogenesis in the Ovarian Follicles of the Goldfish (Carassius auratus) during Gonadotropin-Induced Oocyte Maturation

Both partially purified chum salmon gonadotropin and 17α-hydroxyprogesterone stimulated in vitro production of testosterone by postvitellogenic follicles of goldfish ( Carassius auratus ). Chum salmon gonadotropin further enhanced the conversion of exogenously supplied 17α-hydroxyprogesterone to 17α, 20β-dihydroxy-4-pregnen-3-one. The increased medium concentrations of 17α, 20β-dihydroxy-4-pregnen-3-one were associated with the induction of final oocyte maturation.
The capacity of postvitellogenic follicles to produce steroids in response to exogenous 17α-hydroxyprogesterone was examined in females at various stages of final oocyte maturation following the administration of human chorionic gonadotropin in vivo combined with elevation of holding temperature. The maximum production of testosterone in response to 17α-hydroxyprogesterone was obtained in follicles from initial controls. In contrast, 17α 20β-diOHprog production was very low in initial controls and markedly increased during oocyte maturation (3–6 hr following injection), followed by a significant decrease in follicles collected at 15 hr. Estradiol-17β production by the follicles was very low at any stages of gonadotropin-induced oocyte maturation. These results suggest that gonadotropin-induced shift in the biosynthetic pathway in the follicle from the secretion of predominantly testosterone to 17α, 20β-dihydroxy-4-pregnen-3-one secretion is a prerequisite step for the induction of oocyte maturation in goldfish.     

Role of secreted proteins and gonadotrophins in promoting full maturation of porcine oocytes in vitro

Experiments were designed to identify the extent to which follicle cells and hormones contribute to the developmental competence of porcine oocytes matured in vitro. Oocyte-cumulus complexes were collected from ovaries by dissection and cultured in 2 ml of TCM199-based medium in 5% CO₂ in humidified air at 38.5°C. This basic maturation system was supplemented, for either the first 24 hr only or for the 48-hr culture period, with 1) everted follicle shell alone, 2) gonadotrophic hormones alone, or 3) both follicle shells and hormones. The effect of these treatments was evaluated on 1) meiotic maturation rates, 2) the capacity of matured eggs to undergo activation and early cleavage, and 3) changes to the profile of proteins secreted into the culture medium. The results showed that 1) supplementation with either follicle shell or hormones alone increased the rates of meiotic maturation over the nonsupplemented control group, and 2) combined follicle shell and hormonal supplementation yielded the highest rates for maturation, activation, and cleavage but only when hormonal supplementation was removed after the first 24 hr of culture. Proteins of 30, 37, 45, and 46 kD, but of unknown function, were secreted during the first 24 hr into the culture medium in groups supplemented with follicle shells. The addition of hormones did not affect this pattern of secreted proteins. It is possible that some secreted proteins may act to facilitate full maturation of pig oocytes. Mol. Reprod. Dev. 47:191–199, 1997. © 1997 Wiley-Liss, Inc.     

Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro

The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 μg/ml human follicle-stimulating hormone (hFSH), (2) 10 μg/ml human luteinizing hormone (hLH), (3) 1.0 μg/ml hFSH and 10 μg/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 ± 1.7 hr; hLH: 10.1 ± 1.8 hr; both gonadotropins: 8.8 ± 1.1 hr) when compared to controls (17.4 ± 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 ± 1.2 hr; hLH: 35.1 ± 1.4 hr; both gonadotropins: 35.2 ± 1.3 hr; no gonadotropins: 34.1 ± 1.2 hr). The mean interval to extrusion of PB1 was 34.1 ± 0.6 hr. In conclusion, GVB and PB1 extrusions appear to be, in part, independently regulated events in macaque oocytes matured in vitro since the timing of PB1 extrusion is not tightly coupled with the onset of GVB. Although the developmental potential of oocytes may be enhanced by gonadotropins, alternative approaches must be developed to improve the poor competence of oocytes from nonstimulated monkeys to mature in vitro. © 1994 Wiley-Liss, Inc.     

Induction and Inhibition of Meiotic Maturation in Follicle-enclosed Mouse Oocytes by Forskolin

A continuous exposure of follicle-enclosed mouse oocytes to ovine luteinizing hormone (LH, 10 μg/ml) in vitro resulted in a 3-fold elevation of CAMP levels in the follicle cells, but not the oocytes, with subsequent oocyte maturation. When follicle-enclosed oocytes were exposed to forskolin (0.01–10 μM) for 2 hr and then incubated in forskolin-free medium (transient exposure group), oocytes underwent germinal vesicle breakdown in a dose-dependent manner. In contrast, a continuous exposure of the follicles to forskolin (10 μM) for up to 10 hr failed to induce resumption of meiosis. Follicle cell cAMP levels increased within 2 hr after the initial exposure to forskolin, and thereafter decreased rapidly regardless of whether forskolin treatment was transient or continuous. A similar transient increase in oocyte cAMP levels was observed after transient or continuous treatment with forskolin. It was evident, however, that at any time examined oocyte cAMP levels were consistently higher in the continuous exposure group than in the transient exposure group. Furthermore, a continuous exposure to forskolin also blocked LH-induced meiotic maturation. These findings suggest that elevated levels of cAMP in the oocyte block meiotic maturation in mouse oocytes. The present results further suggest that an increase in follicle cell cAMP levels is essential to the LH-induced meiotic maturation.     

17α,20β,21-Trihydroxy-4-pregnen-3-one: the probable maturation-inducing steroid of the Lusitanian toadfish

17,20β,21-Trihydroxy-4-pregnen-3-one (17,20β,21-P) was identified as the major metabolite of incubations of Lusitanian toadfish Halobatrachus didactylus ovarian follicles with [³H]-17hydroxyprogesterone. The potency of several steroids in inducing germinal vesicle breakdown of follicle-enclosed oocytes of Lusitanian toadfish was systematically examined by using an in vitro germinal vesicle breakdown (GVBD) bioassay. 17,20β-Dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-P, two confirmed maturation-inducing steroids (MIS) in teleosts, were the most potent in inducing GVBD with ED₅₀s ranging between 9 and 271 nM. Structure-activity relationships followed similar patterns to what has been observed in similar bioassays, i.e. a vital requirement for 17- and 20β-hydroxyl groups in C21 steroids and a reduction in activity of 14 and 5–6%, respectively, for 5-pregnene and 5β-pregnanes compared to 4-pregnenes. Corticosteroids, testosterone and 17β-oestradiol were ineffective. Folliculated oocytes stimulated by pituitary homogenate produced 17,20β,21-P from endogenous substrates in amounts one order of magnitude higher than 17,20β-P. These results strongly support the hypothesis that 17,20β,21-P is the likely MIS in this species.     

Polysaccharide Complexes in Full-Grown Oocytes of the Newt, Pleurodeles, and Changes in their Distribution During Progesterone-Induced Maturation

Immature full-grown oocytes of Pleurodeles waltlii contain large amounts of small electron-dense polysaccharidic granules. These granules lack a limiting membrane, and have a dense but heterogeneous matrix and an apparent diameter of 24–36 nm. Their structure, organization and distribution strongly suggest that they are glycogen granules. On the other hand, mature oocytes both after oviposition or 22–24 hr after in vitro progesterone stimulation contain no polysaccharide granules or complexes. During the first 9–10 hr after hormonal stimulation, granules were abundant and present both individually and as large strands occupying most of the space between the organelles. Granules were frequently found packed together and arranged in regularly arrayed stacks within large subcortical ant cortical vacuoles. Between 4 and 6 hr after progesterone addition, oocytes released the contents of vacuoles to the outside. Between about 11 and 14 hr after progesterone addition, oocytes still contained large amounts of polysaccharide complexes, but the vacuoles were empty. From about 15 hr after progesterone treatment until the end of maturation, the complexes progressively disappeared from the cytoplasm, coincident with the detachment of the follicle cell layer from the oocytes and a reduction in the number and size of microvilli.     

In Vivo Study of Vitellogenin-Gold Transport in the Ovarian Follicle and Oocyte of Xenopus laevis

The transport of injected vitellogenin (VTG)-gold in the ovarian follicle and developing oocyte in Xenopus is described. The gold particles reached the extracellular spaces of the theca and interfollicular spaces within 1 and 2 hr, respectively, after a tracer injection at 20°C. The tracers moved through channels between the constitutive cells of both the capillary endothelium and the follicle cell layer.
Compartments in the peripheral cytoplasm of vitellogenic oocytes at stage IV, which relate to yolk formation, seemed to be segregated as follows: (a) internalization compartment consisting of coated pits and vesicles of the oolemma covering the oocyte "macrovilli", (b) transport compartment of endosomes and multivesicular endosomes in the oocyte cortex, and (c) crystallization compartment of primordial yolk platelets (PYP) in the sub-cortical region. The gold particles appeared in the internalization and transport compartments at 3–6 hr after the tracer injection and in the cystallization compartment at 12–18 hr. The VTG, internalized by receptor-mediated endocytosis, was transferred from coated vesicles to multivesicular endosomes by vesicle-to-vesicle fusion. VTG crystallization took place in globular-shaped PYPs of about 1 μm. At 24 hr after the tracer injection, the gold particles appeared in completely crystallized yolk platelets, most of them clustered in the superficial layer and some integrated into the crystals.     

Follicle cell regulation of protein synthesis and developmental competence in sheep oocytes

The developmental capacity of sheep oocytes cultured outside the follicle was greatly increased by the presence of high concentrations of gonadotrophins (10 micrograms/ml) in the medium. However, even under these conditions, the developmental capacity of the oocytes was only half that of oocytes cultured within the intact follicle. The presence of the cumulus was essential for development; nearly all denuded oocytes failed to undergo cleavage. Maturational changes in the oocyte involving increased amino acid uptake increased incorporation and specific changes in protein synthesis were inhibited by the follicle cells; this suppression was alleviated by gonadotrophic hormones. The cumulus cells suppressed amino acid incorporation and, to some extent, the changes in protein synthesis. However, the suppression of amino acid uptake required the presence of the whole follicle. Patterns of protein synthesis by oocytes cultured outside the follicle differed from those in oocytes cultured within the follicle, irrespective of the presence of the cumulus or gonadotrophins. Analysis of single oocytes cultured outside the follicle showed that the protein profiles varied markedly even under identical culture conditions.     

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.     

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.     

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.     

Oocyte size and structure in relation to blood plasma steroid hormones in individually monitored, spawning Atlantic cod

A series of seasonal blood and ovarian tissue samples were taken from five female Atlantic cod Gadus morhua at different times between various batches of eggs and subsequently examined for oocyte size, plasma steroid hormones and ultrastructure of the cortex oocyte cytoplasm and follicle layer. This detailed protocol demonstrated that the spawning cod ovary is vitellogenically very active. Individual oocytes were able to develop at different rates, and at least two cohorts of oocytes with a different developmental status were present simultaneously. The concentration of estradiol-17β varied between batches and decreased over the season. The levels of 17,20β-P and 11-deoxycortisol were generally low. Oocytes of an advanced developing cohort showed numerous coated vesicles and closely apposed oocytic and follicular microvilli located in the chorionic pores. The number of oocytic microvilli was estimated at about 1–8 million cell⁻¹, which increased the oocyte surface area by a factor of 12. Furthermore, the oocytic microvilli were found to stretch during final maturation. This stage-specific mobility of microvilli is discussed in relation to further incorporation of yolk and the relevant actin-based cytoskeleton observed for other classes of animals.