Oogenesis in Fundulus heteroclitus: I. Preliminary observations on oocyte maturation in vivo and in vitro

During the breeding season, the ovary of Fundulus heteroclitus contains follicles of all sizes up to around 1.8 mm in diameter and continuously ovulates eggs (1.7 mm) into the ovarian lumen. Vitellogenic follicles appear to extend up to 1.3 mm, while those larger than 1.4 mm are undergoing hydration concomitant with maturation. If feeding ceases, vitellogenesis appears to stop and maturational stages flush out of the ovary. Refeeding causes reappearance of maturational stages. A culture procedure was developed for dissected F. heteroclitus follicles and a size range (1.3–1.4 mm) was defined which responded to added hormones by undergoing the same maturational changes (germinal vesicle breakdown, fusion, and peripheral detachment of oil droplets, appearance of activability) as occurs in vivo, although the extent of hydration was not quite as extensive. Ovulation, however, did not consistently occur in vitro. Both 11-deoxycorticosterone and progesterone are potent inducers of maturation in vitro, with median effective doses being 10–20 ng/ml. Hydrocortisone is about one-tenth as active. Human chorionic gonadotropin promotes maturational changes in about 25% of the follicles over a broad concentration range (1–100 IU/ml). The environmental and hormonal mechanisms which influence oocyte maturation in teleosts are discussed in relation to our findings for F. heteroclitus.     

Molecular differentiation of the rabbit ovum. I. During oocyte maturation in vivo and in vitro

The normality of nuclear and cytoplasmic maturation of rabbit oocytes, matured in vivo and in vitro, has been assessed by cytogenetic and electrophoretic criteria. The findings indicate not only that nuclear maturation in vivo and in vitro are directly comparable, but also, as observed by high-resolution, two-dimensional polyacrylamide gel electrophoresis, (1) that both qualitative and quantitative changes in the pattern of polypeptide synthesis occur during maturation, (2) that these patterns are directly comparable in oocytes that had been matured either in vivo or in vitro, and (3) that each stage of maturation is associated with the appearance of specific polypeptides in the autoradiographic patterns. The major differences observed between oocytes matured under these two conditions are (1) that several polypeptides fail to appear in in vitro matured oocytes at the time they are detected in vivo and (2) that the synthesis of some polypeptides is prolonged in vitro compared to in vivo matured oocytes.     

Nutrition and fetal brain maturation : II. Impact of maternal starvation on changing levels of acetylcholinesterase and enolase in vitro

The impact of maternal starvation during Days 17-20 of gestation was examined in 20-day fetal rat brain tissue cultured for 6 days in MEM and 10% adult rat serum. Acetylcholinesterase (AChE) activities were consistently greater in fetal brain cell cultures from starved mothers. When fetal tissues from starved mothers were continuously exposed to 72-h fasted serum, AChE activities increased from 1.03 +/- 0.14 to 1.59 +/- 0.21 mumol/h/mg protein (P less than 0.001). In fetal tissues from fed mothers, lower AChE activities were increased from 0.78 +/- 0.09 to 1.04 +/- 0.07 mumol/h/mg protein (P less than 0.05) when 72-h fasted serum was used to replace the fed serum during incubation. When fetal brain cell cultures from fed mothers were exposed for 6 days to graded concentrations of fed serum (2.5-15%), the activities of AChE fell reciprocally from 1.34 +/- 0.10 to 0.82 +/- 0.12 mumol/h/mg protein (P less than 0.05). The levels of AChE activity in tissues exposed to fasted serum were consistently greater, but fell similarly from 1.62 +/- 0.10 to 0.97 +/- 14 mumol/h/mg protein (P less than 0.01), when serum concentrations were increased from 2.5 to 15%. AChE activities were 30% higher in tissues incubated with cycloheximide 10(-3) M (P less than 0.02). Unlike AChE, fetal brain enolase activities were unaffected by maternal starvation. In fetal brain cell cultures from fed mothers, enolase fell from 1.85 +/- 0.10 to 1.37 +/- 0.12 mumol/min/mg protein following exposure to fasted instead of fed serum (P less than 0.02). In fetal cultures from starved mothers, enolase activities were depressed similarly from 1.76 +/- 0.08 to 1.41 +/- 0.09 mumol/min/mg protein when fasted replaced fed serum (P less than 0.02). Thus, the fetal brain cell cultures appear to maintain enzymatic realignments imposed by maternal starvation for at least 6 days. In addition, serum from fasted animals has significant growth inhibiting properties manifested by heightened activities of AChE and lower activities of enolase.     

Effects of in vitro culture of mouse fetal gonads on subsequent ovarian development in vivo and oocyte maturation in vitro

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 µm in diameter and had competence to resume meiosis in vitro . When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences ( P  < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.     

Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome

The potential benefits of assisted reproduction techniques, such as in vitro maturation (IVM) and in vitro fertilization (IVF) in canids, are linked to the protection and saving of species threatened by extinction due to worldwide habitat destruction and pollution. In both domestic and wild species, these technologies will form the basis for the next leap in reproductive performance by improving fertility rates in valuable middle-aged females, by improving pregnancy rate in infertile or sub-fertile populations and by rescuing biological material to replenish populations of endangered species. In vitro techniques are supposed to answer the reproductive questions of canids, to introduce new methods for contraception and to compete with artificial insemination (AI) as the major or predominant method of embryo production, oocyte- and embryo cryopreservation and cloning. The causes affecting in vitro meiosis of dog oocytes are likely to be diverse. Incomplete understanding of the events associated with oocyte developmental competence are imputed to species reproductive physiology, medium composition and source of ovarian oocyte population used for in vitro maturation. This review addresses some issues on the current state of in vitro maturation and in vitro fertilization of canine oocytes.     

In vitro and in vivo maturation of llama oocytes

Cumulus-oocyte complexes (COC) were collected from abbatoir-derived llama ovaries and cultured in vitro for 28, 30, or 36 h at 39 degrees C in 5% CO2 to determine the time required for maturation. The majority of COC (n=298, 87%) were classified as categories 1 and 2 (COC with > or =5 layers or 2-4 compact layers of cumulus cells, respectively) and homogeneous ooplasm, and the proportion that underwent nuclear maturation (MII) was 78, 81 and 80%, after 28, 30 and 36 h, respectively (P=0.65). To compare the effectiveness of FSH versus eCG for inducing in vivo maturation, in experiment 2, llamas (n=20 per group) were treated with: (1) 25 mg FSH, twice-daily for 4 day, plus 5 mg armour of LH at the end of FSH treatment; or (2) 1000 IU of eCG, plus 5 mg armour of LH 4 day after eCG treatment. The FSH- and eCG-treated groups did not differ (P=0.85) with respect to the number of follicles > or =6mm at the time of COC collection (17.9+/-2.2 versus 17.7+/-2.2), the number of COC collected (10.7+/-2.1 versus 11.2+/-2.3 per llama), or the collection rate per follicle aspirated (71 versus 74%). As well, no difference (P=0.49) was detected between the FSH and eCG groups in the number of expanded COC collected (8.3+/-2.1 versus 10.6+/-2.2) or the number of COC at the MII stage (6.9+/-1.8 versus 8.9+/-1.9). In conclusion, llama oocytes reached MII as early as 28 h after in vitro culture and both FSH and eCG were equally effective in inducing ovarian superstimulation. Treatment with LH after either FSH or eCG superstimulation permitted the recovery of a preponderance of expanded COC in metaphase II that may be suitable for in vitro fertilization without in vitro maturation.     

Mitochondrial maturation during neuronal differentiation in vivo and in vitro

The evolution of the mitochondrion has been followed within differentiating neuronal cells, both in primary cultures of neurons from fetal rat cortex and during rat brain cortex maturation. Changes in total mitochondrial proteins (mt-proteins) were evaluated, and qualitative changes in the mt-proteins pattern were analyzed using the Western blot technique. The evolution of mt-protein contents in cultured neurons resembles what is observed during rat brain maturation. The mitochondrion exhibits pronounced changes in the course of neurogenesis, in particular, bursts of mitochondrial masses accompanying the successive steps of neurogenesis are observed. There are indications that protein equipment of mitochondria during neuronal development undergoes variations. Although more work is required to establish the significance of these correlations, the present data might suggest an important role of the mitochondrion in neurogenesis.     

Steroidogenesis of the fetal adrenal gland in vitro: influence of metopirone applied in vivo and in vitro.

In vitro conversion of 4-14C-progesterone into corticosteroids in the adrenal glands of rat fetuses treated with Metopirone (Su 4885) on the last day of intrauterine development was studied. After a 1-hr incubation of the adrenal glands of fetuses injected with Metopirone, hydroxylation of progesterone into corticosterone (B), 18-hydroxycorticosterone (18-OH-B) and 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) decreased and the synthesis of 11-deoxycorticosterone increased. Following preincubation of the fetal adrenal glands and 1-hr incubation with Metopirone, hydroxylation of progesterone into DOC increased and the synthesis of B decreased. Preincubation and a 2-hr incubation with Metopirone caused a decrease in the synthesis of B, 18-OH-B and 18-OH-DOC and an increase in DOC. The results constitute direct evidence of the ability of the fetal adrenal glands to synthesize all corticoids and indicate that most probably corticoids are synthesized by the fetal adrenal glands in the same way as in the adrenals of adult animals.     

Meiotic competence of bovine fetal oocytes following in vitro maturation

The in vitro ability between fetal and cow oocytes to resume meiosis and progression to metaphase-II (M-II) was compared. Cumulus oocyte complexes (COCs) were harvested from 2 to 6 mm follicles from ovaries of 7.5 month to term fetuses and adult cows. Cumulus cells were removed using 3 mg/ml hyaluronidase and repeated pipetting. Denuded oocytes were fixed in 3% glutaraldehyde, stained with DAPI and evaluated under fluorescent microscopy for nuclear status before in vitro maturation (IVM). COCs from fetal and adult ovaries were also matured in 200 microl droplets of medium 199 supplemented with 10 microg/ml FSH, 10/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM hepes and 10% FBS for 24 h at 39 degrees C and 5% CO(2). Matured oocytes were fixed, stained and evaluated as explained above for nuclear status namely stage of germinal vesicle (GV) development and subsequent meiotic competence. Data were analyzed using chi-square analysis. The majority of fetal oocytes (P<0.05) before IVM were at GV stages GV-I (27.7%), GV-II (37.6%) and GV-V (22.8%) compared to cow oocytes, which were at GV stages IV (28.3%) and V (46.7%). After IVM, fewer fetal oocytes were at earlier stages of GV development and majority (P<0.05) were at GV-V (24.0%), premetaphase (17.4%) and metaphase-I (M-I: 7.2%) stages. However, after IVM, more cow oocytes matured to M-II than did fetal oocytes (93.7% versus 26.9%; P<0.05). In conclusion, fetal oocytes do not mature in vitro as well as cow oocytes. Our findings suggest that the low meiotic competence of fetal oocytes can be attributed to their being at earlier stages of GV development before IVM.     

GABA, a key transmitter for fetal brain maturation

GABA, the principal inhibitory transmitter excites immature neurons in all animal species studied. This is due to the higher intracellular concentration of chloride at early developmental stages. Excitatory actions of GABA play an important action in brain maturation. Recent observations also suggest an abrupt shift during delivery that exerts a neuro-protective action contributing to reduce the sequels of trauma and anoxic episodes. These observations have important clinical implications in relation to delivery associated insults but also preterm delivery and more generally consumption of agents during gestation.     

Tetrahydropapaveroline: formation in vivo and in vitro in rat brain

Gas chromatographic-mass spectrometric techniques have been used for the measurement of tetrahydropapaveroline (THP) from brain. The formation of THP from 1-DOPA or dopamine $\text{in vitro}$ has been confirmed by obtaining a complete mass spectrum of the product as its trifluoroacetylated derivative. Following chronic administration of 1-DOPA or 1-DOPA in combination with ethanol to rats, THP formed $\text{in vivo}$ in in brain could be detected in small quantities although it could not be detected when ethanol alone was administered.     

Factors involved in vivo and in vitro maturation of canine oocytes

The domestic dog could be a valuable model for studying and developing assisted reproduction in taxonomically related endangered Canids. However, the efficiency of in vitro oocyte maturation is very low in this species compared to that of other mammalian species and this limits the development of reproductive biotechnologies, such as in vitro embryo production, cryopreservation, or nucleus transfer. In canine species the female gamete has unique characteristics: the oocyte is exposed to high concentration of progesterone in the follicular environment, it is ovulated in the dictyate state, and resumes and completes meiosis in the oviduct. Therefore, optimum conditions for in vitro maturation of dog oocytes may differ from other mammalian models in which follicles, where estrogens are the dominant hormones, ovulate oocytes at the Metaphase II stage of the first meiotic division. An in vitro culture system needs to be based on in vivo conditions in order to create a microenvironment similar to that in which oocyte development occurs physiologically, but little is known on mechanisms regulating oocyte maturation in the dog. This review analyzes the known factors involved in canine oocyte maturation in vivo and in vitro in order to suggest on which aspects future investigations may be focused.     

Changes in protein synthesis during maturation of sheep oocytes in vivo and in vitro.

The qualitative profiles of the proteins synthesized by sheep oocytes at various stages of maturation were determined by electrophoretic separation in one dimension on polyacrylamide SDS gels. No change in protein synthetic pattern was observed in ooce changes had taken place in at least 12 separate protein bands. Marked alterations in the synthesis of some proteins were apparent 15 h after LH; formation of proteins in 5 of the original bands was either reduced or not detectable, while new synthesis was evident from the appearance of 7 additional bands. The pattern of proteins produced by oocytes cultured within the follicle corresponded closely with that observed in vivo: changes in synthesis were initiated about 9 h after addition of gonadotrophin and were completed by 15 h. Oocytes cultured outside the follicle in a gonadotrophin-containing medium did not exhibit a change in protein synthesis and at 15 h only those proteins produced during the early stages of maturation were being synthesized.     

Comparative maturation of cynomolgus monkey oocytes in vivo and in vitro

Background  

In vitro maturation (IVM) of oocytes followed by fertilization in vitro (IVF) and embryo transfer offers an alternative to conventional IVF treatment that minimises drug administration and avoids ovarian hyperstimulation. However, the technique is less efficient than maturation in vivo. In the present study, a non-human primate model was used to address the hypothesis that the number of oocytes is increased and their nuclear and cytoplasmic maturity after IVM are improved when maturation is initiated in vivo by priming with hCG.     

The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo.

Inhibition of nitric oxide (NO) synthase activity by L-NG-Nitroarginine (NO2Arg) in brain preparations is not reversed by dialysis and is enhanced by prolonged preincubation of NO2Arg with the enzyme. By contrast, the weaker inhibition by NO2Arg of macrophage NO synthase is fully reversible. NO2Arg inhibits NO synthase activity in the brain after i.p. administration of 5 or 50 mg/kg. This in vivo inhibition also appears to be irreversible. The potent in vivo inhibition of central NO synthase by NO2Arg may facilitate studies of the physiologic function of NO as a neuronal messenger.     

Hizikia fusiforme extract enhances dendritic cell maturation in vitro and in vivo

ABSTRACT

Dendritic cells (DCs) are play critical roles in the priming and regulation of immune responses. DCs rapidly process and convey these antigens to prime antigen-specific T cells. Therefore, regulation of DCs functions is important for immunity and immunotherapies. Immune adjuvants for DCs activation are needed to improve the efficacy of vaccines against tumors and many infectious diseases. Therefore, we demonstrate that H. fusiformis extract can regulate DCs maturation and activation. H. fusiformis extract induced costimulatory molecules (CD 80 and CD86), antigen-presenting molecules (major histocompatibility complex (MHC) I and II), CCR7 expression, and interleukin (IL)-12 production in DCs. These effects are associated with upregulation of mitogen-activated protein kinase (MAPK) signaling pathway. In addition, H. fusiformis extract induces costimulatory molecules on splenic DCs and activated CD8⁺ T cells in vivo. Taken together, these findings suggest that H. fusiformis extract may be a potential efficient immune therapeutic compound in DCs-mediated immunotherapies.