Use of a specific aromatase inhibitor for determining whether there is a role for oestrogen in follicle/oocyte maturation, ovulation and preimplantation embryo development

The specific role of oestrogen in follicular maturation, ovulation and early embryonic development was investigated using Fadrozole (CGS 16949A), a non-steroidal aromatase inhibitor, to block oestrogen synthesis specifically and effectively in experimental animals. Induced and normal cyclical follicular maturation as well as normal and hCG/LH-induced ovulation were relatively unaffected by significantly depleting oestrogen in all animals (hamsters, rabbits, monkeys) studied other than rats. Fadrozole treatment significantly reduced the number of healthy antral follicles produced and the ovulatory response to exogenous hCG of immature rats primed with pregnant mares' serum gonadotrophin. The effect was specific, in that exogenously administered oestrogen reversed the blockade. Depletion of oestrogen, starting early in pro-oestrus in hamsters, had no effect on ovulation, oocyte maturation and fertilization, as normal implantation sites were seen on day 6 after coitus. In rabbits, oestrogen depletion during the periovulatory phase affected oviductal morphology and function. Although fertilization was not impaired, early embryo development did not appear to be normal. In monkeys, oestrogen depletion during the follicular phase did not lead to a block of follicular maturation or ovulation but resulted in a significant reduction in secretion of cervical mucus. Administration of either Fadrozole or Tamoxifen during the early luteal phase in cyclic monkeys that were allowed to mate prevented implantation and this appears to be due to impaired fertilization or faulty embryo development. These results suggest that, although there is a clear requirement for oestrogen to support the reproductive cycle in the female, the need for oestrogen in regulating specific events is species dependent.

CORRELATION OF ZONA-BINDING WITH OOCYTE MATURATION AND SPERM MOTILITY IN RHESUS-MONKEYS BY HEMIZONA ASSAY

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PB1). The mean numbers of sperm bound to hemizona for PBI, PVS, GV, and MC groups were 132.9 +/- 12.0, 71.5 +/- 10.1, 36.1 +/- 4.0, and 20.1 +/- 2.9 (Mean +/- SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 +/- 2.2 and 25.3 +/- 2.9 (Mean +/- SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1)The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the ''zona maturation'' hypothesis. (C) 1994 Wiley-Liss, Inc.

A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish

Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca2+-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development. (C) 2003 Elsevier Inc. All rights reserved.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.

Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone I-Il as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longer-lasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

INVOLVEMENT OF CA(2+) SIGNALING PATHWAY DURING OOCYTE MATURATION OF THE NORTHERN QUAHOG MERCENARIA MERCENARIA

In many molluses, it has been found that Ca2+ signaling pathway is involved in the resumption of meiotic maturation in oocytes. To better understand the possible role of Ca2+ signaling pathway in regulating meiotic maturation in oocytes of the northern quahog Mercenaria mercenaria, free extracellular Ca2+, A23187 (calcium ionophore), verapamil (calcium channel blocker), and trifluoperazin (calmodulin antagonist) were used to incubate oocytes or serotonin-induced oocytes by pharmacological methods. Results show that extracellular Ca2+ (50 similar to 200 mM) and A23187 (1 similar to 10 mu M) can stimulate the meiotic maturation. In addition, verapamil (1 similar to 100 mu M) and trifluoperazin (10 similar to 1,000 mu M) could inhibit serotonin-induced oocyte maturation. Therefore, Ca2+ is essential for the reinitiation of meiotic maturation in oocytes of the northern quahog. Moreover, an increase i [Ca2+]i can promote meiotic maturation.

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.

Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice

Background: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. Methods: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. Results: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. Conclusions: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis

17β-estradiol, 17α-20β-dihydroxy-4-pregnen-3-one (17α-20β-P), and testosterone levels were measured in plasma samples obtained from vitellogenic coho salmon (Oncorhynchus kisutch) before and 32 days after injection of the aromatase inhibitor Fadrozole (AI). Plasma 17β-estradiol levels decreased significantly 6 h after injection in all AI treated fish. The higher the dose the longer the maintenance of low plasma 17β-estradiol levels. Inversely, plasma 17α-20β-P increased significantly 6 h after injection in all AI treated fish, and the higher the dose the longer the maintenance of high plasma 17α-20β-P levels. At 48 h after injection plasma testosterone levels were significantly higher in the AI treated groups. The oocyte maturation index showed that multiple injections with AI retarded oocyte development. Besides, oocyte diameter and GSI were lower in the same group, which presented high incidence of atresia of vitellogenic oocytes. The ovarian follicles and brain of the fish which received multiple injections secreted less 17β-estradiol, in vitro. These findings suggest that aromatase inhibitors such as Fadrozole may have a potential as a tool to regulate sexual development in salmon.

Spawning failure in Brycon amazonicus may be associated with ovulation and not with final oocyte maturation

Avaliaram-se os possíveis mecanismos envolvidos com a falha na desova de matrinxãs (Brycon amazonicus), submetidas à indução hormonal por extrato bruto de hipófise de carpa. Para tal, após a extrusão, os ovários foram coletados e analisados histomorfometricamente. Nas fêmeas que não desovaram (FNDs), a maioria dos ovócitos vitelogênicos remanescentes nos ovários atingiu a maturação final, apresentando quebra de vesícula germinativa, mas não foram ovulados (NOs). Consequentemente, estas fêmeas apresentaram frequências mais baixas de folículos pós ovulatórios (5%) quando comparadas com a que desovou (FD) (23%). Com relação aos NOs, os valores se inverteram e a frequência destes nas FNDs (21%) foi maior do que na FD (3%). Estes dados indicam que as falhas na desova desta espécie estão provavelmente relacionadas com a ovulação, uma vez que a maturação final dos ovócitos ocorre de forma similar tanto nas FNDs como na FD. Os dados sugerem que as substâncias que promovem a ovulação, como as prostaglandinas, podem aumentar o sucesso de desova em peixes reofílicos.

Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

Avaliaram-se o efeito do IGF-I na maturação in vitro (MIV) (experimento I) e no desenvolvimento embrionário (DE) (experimento II) de oócitos bovinos fecundados in vitro, quanto às taxas de clivagem (TC), de blastocistos (TB) e de eclosão (TE). Para MIV, complexos cumulus-oócitos imaturos foram cultivados em meio TCM-199 suplementado com HEPES, bicarbonato e piruvato de sódio, aditivos, soro fetal bovino (meio B-199) e gonadotrofinas 14U/ml de PMSG e 7U/ml de hCG). Para o desenvolvimento embrionário, os oócitos/zigotos foram cultivados em meio B-199 com células epiteliais do oviduto bovino em suspensão sob óleo de silicone. As condições de cultivo in vitro para ambos os experimentos seguiram os tratamentos: 1- meio B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. Todas as culturas foram realizadas a 38,5° C em atmosfera com 5% de CO2 e os dados foram analisados pelo teste do qui-quadrado. No experimento I, não houve diferença (P>0,05) entre os tratamentos quanto às TC, TB e TE, quando o meio de MIV foi suplementado com IGF-I. No experimento II, a adição de IGF-I ao meio de DE resultou em aumento na TC (P<0,05) mas não influenciou a TB e a TE. A adição de 200 ng/ml de IGF-I ao meio DE melhorou a TC (71,1%) quando comparada com a TC dos grupos de 100 ng/ml de IGF-I (57,6%) ou controle (56,7%), entretanto não houve diferença quando comparada com a dos grupos de 50 ng/ml (69,4%) ou 10 ng/ml (73,1%) de IGF-I. Não houve efeito benéfico na adição de 10 a 200 ng/ml de IGF-I nos meios de MIV e de DE com relação ao desenvolvimento de embriões produzidos a partir de oócitos maturados e fecundados in vitro.

The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ovary and oocyte maturation of the tick Amblyomma brasiliense Aragao, 1908 (Acari: Ixodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)