Hormonal dissociation of ovulation and maturation of oocytes: Ovulation of immature amphibian oocytes by prostaglandin

Prostaglandin involvement in ovulation and maturation of amphibian (Rana pipiens) ovarian follicular oocytes was investigated using in vitro-cultured ovarian follicles. Exposure of follicles to PGF₂α during culture stimulated variable but generally low levels of ovulation without concomitant induction of maturation. Addition of PGF₂α to cultured follicles markedly enhanced the incidence of ovulation in follicles exposed to progesterone or frog pituitary homogenate (FPH). Onset of the ovulatory process was further accelerated following addition of PGF₂α to FPH-treated follicles. PGE, in contrast to PGF₂α, exhibited no stimulatory effects on ovulation and consistently inhibited ovulation induction by FPH and progesterone. Cytological analysis of follicles undergoing ovulation revealed that ovulation of immature oocytes induced by PGF₂α varied markedly from that seen following FPH or progesterone stimulation of follicles in vivo or in vitro. Immature oocytes in contrast to maturing oocytes were typically ovlulated with follicle cells still attached to the vitelline membrane. The observations indicate that PGF₂α effected follicle rupture and contraction of the follicular epithelium and theca without prior separation of the follicle cells from the oocyte. Selective inhibitors of steroid synthesis (cyanoketone) and protein synthesis (cycloheximide) inhibited FPH-induced ovulation and maturation. PGF₂α reversed the inhibitory effects of cyanoketone and cycloheximide on FPH-induced ovulation but not maturation of oocytes. Neither prostaglandins alone or in combination with progesterone or FPH induced ovulation of oocytes following removal of the follicular epithelium. Ovulatory effects of PGF₂α appear to be mediated through the follicular epithelium. Results indicate that ovulation and maturation of amphibian oocytes can be induced independently of each other by separate classes of hormones. Normal synchronization of ovulation and maturation of oocytes may require the combined action of prostaglandins and steroids acting within different follicular compartments.     

Isolation and characterization of 23 microsatellite loci in the yellow tang, Zebrasoma flavescens (Pisces: Acanthuridae)

Twenty‐three microsatellites were isolated from the yellow tang (Zebrasoma flavescens), an ecologically and commercially important reef fish. Genetic diversity was assessed in 90 adults collected from Honokohau, Hawaii. The number of alleles per locus varied from four to 29 (mean = 13.8) and observed and expected heterozygosities ranged from 0.15 to 0.94 (mean = 0.70) and from 0.29 to 0.93 (mean = 0.81), respectively. Eight loci exhibited significant departure from Hardy–Weinberg equilibrium due to the presence of null alleles. Exact tests showed no evidence of genotypic disequilibrium between loci. Overall, loci were well resolved, easy to score and highly polymorphic.     

Role of prolactin in nuclear maturation and ovulation in amphibian oocytes

The present study was undertaken to determine the effect of prolactin (Prl) on Bufo arenarum oocyte maturation and ovulation, two characteristic events of the breeding period, the stage of the sexual cycle in which gamete growth is complete. We observed that Prl, at the doses assayed, did not affect nuclear maturation per se. In addition, when follicles were pretreated with Prl and progesterone was later added to the medium as a physiological nuclear maturation inducer, the percentage of germinal vesicle breakdown obtained with the steroid was unaffected by Prl. The analysis of the in vitro ovulation process demonstrated that pituitary homologous homogenate (PHH) produced a dose- and month-dependent stimulating effect. The maximum percentage of ovulated oocytes was obtained from the end of July to October, the period in which oviposition naturally occurs. Prl by itself did not affect the ovulation process, but when both the hormone and PHH were present in the incubation medium, a significant increase in ovulated oocytes was observed. The results suggest that Prl does not participate in oocyte maturation; however, its presence in the incubation medium would increase oocyte sensitivity to the action of the physiological ovulation inducers.     

Effects of mineral supplements on ovulation and maturation of dog oocytes

The aim of this study was to assess the effects of trace mineral supplements near the time of ovulation on the number of ovulated oocytes, in vivo oocyte maturation and pregnancy for dog cloning. Sixteen oocyte donor dogs were used in each control and mineral supplement group, and 136 and 166 corpora lutea were counted from each group. No significant difference was observed between oocyte recovery rates in the control (91.2 ± 2.7%) and mineral (89.9 ± 2.7) groups. Proportions of mature (86.2 ± 7.2 and 88.4 ± 6.8%) and aged (13.8 ± 7.2 and 11.6 ± 6.8%) oocytes were not different in the control and mineral groups, respectively. Oocytes with fair (91.5 ± 3.6 and 93.6 ± 2.1%) and poor (8.5 ± 3.6 and 6.4 ± 2.1%) quality also showed no difference between the control and mineral groups. The concentrations of manganese and ferrous iron were higher and lower on the day of ovulation, respectively, in both groups, but trace element concentrations in peripheral blood were not affected by mineral treatment. Oocytes were used to make cloned embryos; after embryo transfer, four and two pups were delivered from the control and mineral group, respectively, but there was no difference in the delivery rate (4.6 and 2.7%). In conclusion, intravenous mineral supplements administered once close to the LH surge in oocyte donor dogs and recipients had no effect on the number of ovulated oocytes, in vivo oocyte maturation or pregnancy in dog cloning in this study.     

Hormonal stimulation of maturation and ovulation,gamete morphology,and raising of larvae in <Emphasis Type="Italic">Dascyllus trimaculatus</Emphasis> (Pomacentridae)

The experiments on hormonal stimulation of the maturation and ovulation of oocytes of Dascyllus trimaculatus (Pomacentridae) are carried out. Double injections of surfagon (LH-RH-a) (5 + 15 μg/kg of fish body weight) with (or without) the addition of eglonil (5 + 15 mg/kg) are used, and the interval between the injections ranges from 12 to 17 h. Ovulation is registered 33.5–42.0 h after injection I. Morphological changes in oocytes are followed during the process of their maturation. The ultrastructure of spermatozoa and oocyte envelopes is studied. The quality of ovulated oocytes is assessed after their in vitro storage at 25 and 5°C. Preliminary results on the transition of the larvae to exogenous feeding are obtained.     

The behavioral ecology of three Indian Ocean surgeonfishes (Acanthurus lineatus,A. leucosternon and Zebrasoma scopas): their feeding strategies,and social and mating systems

Synopsis The relationship between the morphology, feeding strategies and social and mating systems of three surgeonfishes was investigated. Adults of each defend feeding territories, intra-and interspecifically. The largest species, because of its morphological limitation, relies on food that has to be defended against many other species. It forms large colonies in which fishes singly defend small territories containing high standing crop algal mats. Colony formation is a mechanism by which the efficiency and effectiveness of interspecific territory defense is increased. The smallest species, because of its morphological adaptations, is able to rely most on food that other species cannot efficiently exploit. It forms pairs that defend large territories containing a thin algal mat. It is restricted to the poorest quality habitat by the aggressive activities of more dominant species. The third species, which also forms pairs, has an intermediate feeding strategy. The local coexistence of these three and other surgeonfishes results from a combination of (i) their partitioning both habitat and food resources, and (ii) the populations of two of the most dominant species apparently being below the carrying capacity. Territoriality and the absence of parental care facilitates pair formation in surgeonfishes. Permanently territorial species usually form pairs. The colonial species does not form pairs because the colonial habit facilitates interference of males in each other's spawnings.     

Insulin-transferrin-selenium (ITS) improves maturation of porcine oocytes in vitro

The objective of this study was to determine if insulin-transferrin-selenium (ITS) promoted a nuclear and cytoplasmic maturation of porcine oocytes that better supports subsequent embryonic development. The rate of oocyte in vitro maturation (IVM) in an experimental group treated with hormones for 42 h was significantly increased compared with that in a control group without hormone treatment (47.8% vs. 11.7%, respectively, p < 0.05). Following reduction of the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormone treatment (44.8%), the rate of oocyte IVM was still higher than that of the control group (p < 0.05). To improve porcine oocyte nuclear maturation, 1% ITS was added to medium supplemented with hormones. The rate of nuclear maturation in the ITS-treated group was significantly higher than in the ITS-untreated group (78.6% vs. 54.4%, respectively, p < 0.05). ITS treatment also significantly reduced the per cent of oocytes with type I and type III cortical granule (CG) distribution, respectively, and significantly increased the per cent of oocytes with type II CG distribution (85.3%). These observations indicated that the synchronization rates of nuclear and ooplasmic maturation reached 67.04% (78.56 × 85.33%). In conclusion, the combination of modified Tissue Culture Medium-199 (mM199) + 10 ng/ml epidermal growth factor (EGF) + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulating hormone (FSH) + 1% ITS is suitable for culturing porcine oocytes in vitro, and effectively enhances porcine oocyte nuclear and cytoplasmic maturation.     

Effects of pH on in vitro ovulation of goldfish (Carassius auratus) oocytes

The in vitro effects of pH on goldfish (Carassius auratus) ovulation were investigated. Final oocyte maturation and follicular detachment were induced in vivo in gravid goldfish by HCG injections and elevated holding temperatures. Females were biopsied to determine the time of germinal vesicle breakdown and when oocytes should be removed for in vitro incubations. Prior to ovulation, the ovaries were removed, dissected and mature intrafollicular oocytes were incubated in media of varying pH (7.3-8.9). There was a significant increase in ovulation as the pH of the incubation increased and this ovulation could be blocked by indomethacin. Indomethacin did not inhibit the actual mechanism of oocyte expulsion since exogenous PGF2 alpha induced ovulation in indomethacin blocked incubates. Increased pH did not increase the ovulatory response observed with exogenous PGF2 alpha. The combined results suggest that an increase in incubation pH stimulates prostaglandin synthesis that, in turn, stimulates ovulation.     

Computer-aided meiotic maturation assay (CAMMA) of zebrafish (danio rerio) oocytes in vitro

We have developed a new technique called Computer-Aided Meiotic Maturation Assay (CAMMA) for imaging large arrays of zebrafish oocytes and automatically collecting image files at regular intervals during meiotic maturation. This novel method uses a transparency scanner interfaced to a computer with macro programming that automatically scans and archives the image files. Images are stacked and analyzed with ImageJ to quantify changes in optical density characteristic of zebrafish oocyte maturation. Major advantages of CAMMA include (1) ability to image very large arrays of oocytes and follow individual cells over time, (2) simultaneously image many treatment groups, (3) digitized images may be stacked, animated, and analyzed in programs such as ImageJ, NIH-Image, or ScionImage, and (4) CAMMA system is inexpensive, costing less than most microscopes used in traditional assays. We have used CAMMA to determine the dose response and time course of oocyte maturation induced by 17alpha-hydroxyprogesterone (HP). Maximal decrease in optical density occurs around 5 hr after 0.1 micro g/ml HP (28.5 degrees C), approximately 3 hr after germinal vesicle migration (GVM) and dissolution (GVD). In addition to changes in optical density, GVD is accompanied by streaming of ooplasm to the animal pole to form a blastodisc. These dynamic changes are readily visualized by animating image stacks from CAMMA; thus, CAMMA provides a valuable source of time-lapse movies for those studying zebrafish oocyte maturation. The oocyte clearing documented by CAMMA is correlated to changes in size distribution of major yolk proteins upon SDS-PAGE, and, this in turn, is related to increased cyclin B(1) protein.     

The state of gametes and the type of maturation of follicular oocytes in rats neonatally androgenized by LH-RH-induced ovulation

The character of follicular oocyte maturation and the state of gametes in androgenized rats was studied after LH-RH stimulation. The induction of ovulation with LH-RH has been shown to increase the number of rats with nonovulatory follicules and reduce the number of oocytes maturing prior to the onset of metaphase II. After the induction of ovulation the rate of chromosomal aberrations became higher than in intact animals. Ovulated oocyte population was characterized by the increased number of degenerated gametes. This increase was paralleled by decreased incidence of chromosomal aberrations in ovulated cells.     

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes.

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.     

Patterns of lineage diversification in the genus Naso (Acanthuridae)

The evolutionary history of the reef fish genus Naso (F. Acanthuridae) was examined using a complete species-level molecular phylogeny of all recognized (19) species based on three loci (one nuclear ETS2 and two mitochondrial 16S, cyt b). This study demonstrates that distinct foraging modes and specialized body shapes arose independently at different times in the evolutionary history of the genus. Members of the subgenus Axinurus, characterized by a scombriform morphology, caudal fin structure and pelagic foraging mode, were consistently placed basal to the remaining Naso species, suggesting that pelagic foraging is plesiomorphic and benthic foraging derived in this genus. We used a genus-level phylogeny (nuclear marker, ETS2), which included several taxa from all other acanthurid genera, to obtain a range of age estimates for the most recent common ancestor of the genus Naso. These age estimates (range of 52-43.3 MY) were then used to estimate divergence times (by nonparametric rate smoothing method) of the node giving rise to extant Naso species using the combined sequence data (from all loci). The reconstruction of the pattern of divergence of extant species indicates two sequences of events. The basal species characterized by pelagic foraging modes arose during the Eocene and Oligocene. Most of the remaining Naso species, including those characterized by benthic foraging, arose over a period of 20 MY during the Miocene. Diversification during this period was associated with major plate tectonic and glaciation events, resulting in changes in sea level, ocean temperature and productivity regimes. Regardless of the foraging mode exhibited, all species of Naso have a caudal propulsive unit similar to that observed in pelagic scombriform fishes, a legacy of the basal position of the subgenus Axinurus in the phylogeny of the genus.     

Chronological and ultrastructural changes in camel (Camelus dromedarius) oocytes during in vitro maturation

Cumulus-oocyte complexes (COCs) were collected from non-pregnant camels at a local slaughterhouse by aspiration from antral follicles (2-6 mm). In Experiment I, camel COCs (n=304) were matured in vitro in Hams-F10, fixed at different time intervals (6, 12, 18, 24, 30, 36, 42, or 48 h) and stained with 1% aceto-orcein to assess nuclear changes in culture. A majority of the oocytes (81.5%) underwent germinal vesicle break down (GVBD) between 6 and 12h. Forty-eight percent of the oocytes were observed at the metaphase I (M I) stage by 18 h culture. The percentage of matured oocytes (M II stage) at 30 and 42 h were 66.5 and 71% respectively, which were significantly (p<0.05) different to that observed at 24 h (42.5%). In Experiment II, after different periods of culture (12, 24, 36, or 48 h), the COCs (n=26) were processed for transmission electron microscopy. Expansion of both the cumulus and corona radiate cells occurred between 12 and 24 h in the majority of oocytes concomitant with enlargement of the cumulus cell process endings (CCPEs) in the developed perivitelline space. After 12 h of culture disruption of the junctions between CCPEs and the oolemma was observed together with and breakdown of the GV. For 24-36 h of culture cortical granules had spread and aligned along the oolemma. Signs of degeneration in the cytoplasmic organelles of the oocytes were also observed from less than 36 h. After 48 h of culture, larger vesicles and lipid droplets had appeared in the central part of the oocytes and showed uneven distribution throughout the ooplasm. Predominantly non-penetrating CCPEs were also observed in four oocytes by 48 h. In conclusion, based on both light and electron microscopic evaluations, the optimal culture time for the development of competent Camelus dromedarius oocytes in vitro appears to be 30 h using Hams-F10 medium.     

Morphological criteria of egg quality in marine fishes: Activation and cleavage of eggs of <Emphasis Type="Italic">Zebrasoma scopas</Emphasis> (Acanthuridae)

Morphological changes of the eggs of Zebrasoma scopas obtained just after ovulation and after the storage of ovulated oocytes in the ovarian cavity of the female (in vivo) or in the external medium (in vitro) are described. The dynamics of cortical reaction is followed in noninseminated oocytes, as well as in inseminated oocytes of various quality. The average proportions of eggs exhibiting normal cleavage in the control series (comprised the eggs inseminated just after ovulation) and in the series stored in vivo for 2 and 4 h at 27°C are 83, 30, and 35%, respectively. The average proportions of eggs with normal cleavage in the control series and in the series stored in vitro for 2 and 4 h at 25°C are 88, 79, and 34%, respectively. The storage of oocytes in vitro at 7°C for 2 h leads to the abrupt decrease of their quality with the proportion of normally cleaved eggs reaching only 15%. The types of abnormal cleavage leading to subsequent degradation of cells of the blastodisc and total mortality of eggs are as follows: (1) mitotic cleavage of nuclei accompanied by incomplete cytotomy, (2) mitotic cleavage of nuclei without cytotomy, (3) a weak adhesion between cells, and (4) desynchronization of cell divisions. The methods for the assessment of egg quality just after ovulation and at initial developmental stages are suggested for marine teleost fishes. Published in Russian in Voprosy Ikhtiologii, 2008, Vol. 48, No. 4, pp. 537–552. The article was translated by the authors.     

Evidence for vitellin maturation within oocytes of Perinereis cultrifera (Polychaete annelid)

• 1.1. The relationship between the five native forms of vitellin (V₁-V₅) previously identified in Perinereis cultrifera oocytes was studied by following the distribution of [³H]leucine between these vitellin forms after in vivo injection of the amino acid in young and submature females.
• 2.2. By native polyacrylamide gel electrophoresis and fluorography, the highest molecular weight form of vitellin (V₁, 530,000) was first (one day incubation period) detected as being radioactive while for long incubation periods (3 to 14 days) a progressive shift in labelling was observed from V₁ into the smaller forms of vitellin (V₂, 500,000; V₃, 470,000; V₄, 430,000 and V₅, 390,000).
• 3.3. During the increasing incubation periods, a progressive shift in labelling from a single high molecular weight polypeptide (P₁, 176,000) into lower molecular weight fragments characteristic of the mature vitellin form V₅ was found when the vitellin fractions obtained by immunoprecipitation were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and fluorography.
• 4.4. These results confirm the hypothesis of a precursor-product relationship between V₁ and V₅ via three intermediate forms (V₂ to V₄) and strongly suggest that the conversion process within the oocyte involves progressive proteolytic cleavages of a single precursor component, or polypeptide subunit of V₁, into those that make up V₅, the mature form of vitellin.

     

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes were studied using a new method that allows a clearer visualization of both nucleolus and chromatin after Hoechst staining. The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on nucleolus and nuclear membrane disappearance. While the GV1 to 4 configurations were similar to those reported by previous studies, the GV0 configuration was distinct by the diffuse, filamentous pattern of chromatin in the whole nuclear area. Most of the oocytes were at the GV0 stage in the <1 and 1-1.9 mm follicles, but the GV0 pattern disappeared completely in the 2-2.9 and 3-6 mm follicles. As follicles grew, the number of oocytes with GV1 configurations increased and reached a maximum in the preovulatory follicles 4 hr post-hCG injection. During maturation in vivo, the number of GV1 oocytes decreased while oocytes undergoing GVBD increased. The percentage of oocytes with GV3 and GV4 configurations was constant during oocyte growth except at the 2-2.9 mm follicle stage, but these configurations disappeared completely after hCG injection. On the contrary, the in vitro maturing oocytes showed a large proportion of GV3 and GV4 configurations. There was no significant difference in distribution of chromatin configurations between the nonatretic and atretic follicles, and between oocytes with more than two layers of cumulus cells and those with less than one layer or no cumulus cells. Overall, our results suggested that (i) the GV0 configuration in porcine oocytes corresponded to the "nonsurrounded nucleolus" pattern in mice and other species; (ii) all the oocytes were synchronized at the GV1 stage before GVBD and this pattern might, therefore, represent a nonatretic state; (iii) the GV3 and GV4 configurations might represent stages toward atresia, or transient events prior to GVBD that could be switched toward either ovulation or atresia, depending upon circumstances; (iv) the in vitro systems currently used were not favorable for oocytes to switch toward ovulation (or final maturation); (v) the number of cumulus cells was not correlated with the chromatin configuration of oocytes, indicating that the beneficial effect of cumulus cells on oocyte maturation and development may simply be attributed to their presence during in vitro culture.     

Meiotic maturation of vitrified immature chousingha (Tetracerus quadricornis) oocytes recovered postmortem

The ability to recover and cryopreserve oocytes from postmortem ovaries of endangered or wildlife species holds tremendous potential for conservation using assisted reproductive technologies. The objective of this study was to assess the in vitro meiotic maturation of chousingha (four-horned antelope) oocytes following vitrification using open pulled straw (OPS) method. The average number of oocytes recovered per ovary was 65.6. The proportion of oocytes that matured was significantly lower in vitrified oocytes (29.4%) when compared with fresh oocytes (69.3%). The study provides evidence that it is possible to cryopreserve immature oocytes by vitrification collected from the ovaries of chousingha at postmortem and also demonstrates that these cryopreserved oocytes retain their potential to undergo in vitro meiotic maturation.