Physiological aspects of oogenesis in two species of sticklebacks, Gasterosteus aculeatus L. and Apeltes quadracus (Mitchill)

During May at Woods Hole, female Gasterosteus aculeatus periodically produce clutches of 112±19 eggs with an average diameter of 1·31±0·05 mm. The fish generally have primordial follicles ranging up to 0·56 mm diameter and a clutch of larger follicles undergoing synchronous growth. The size of oocytes within a growing clutch appears to be random within a population, which indicates that recruitment of clutches is not induced by a local environmental event. The largest oocytes within the population of primordial follicles have just begun vitellogenesis but are temporarily arrested. All oocytes within follicles larger than 1·1 mm diameter undergo spontaneous maturation and enlarge to preovulatory size when incubated at 16° C in a simple saline medium. Added deoxycorticosterone can induce similar events in somewhat smaller follicles placed in culture. Thus, in vivo , follicles grow from 0·56 to 1·1 mm diameter by vitellogenesis, and further enlargement is achieved by hydration during steroid-induced maturation. Females carrying follicles in maturational stages also have a new clutch of follicles entering vitellogenic growth from the population of primordial follicles. Injection of human chlorionic gonado-tropin causes a recruitment of follicles into vitellogenesis regardless of the stage of follicles within the growing clutch. One interpretation of these results is that when vitellogenic follicles reach a diameter of 1·1 mm a surge of gonadotropin(s) induces the follicle cells to release steroid, which results in oocyte maturation; the same surge also recruits a new clutch of vitellogenic oocytes. Qualitatively similar results were obtained for Apeltes quadracus ; thus the recruitment phenomenon observed for G. aculeatus may be a general feature among sticklebacks.     

RNA synthesis in the ovary and oocytes of the starfish

Qualitative studies on the in vitro uptake and incorporation of tritiated uridine into RNA of the somatic and germinal elements of the starfish ovary were carried out prior to and during hormone-induced oocyte maturation and spawning.Autoradiography of nonhormone-treated ovaries indicated that the outer ovarian wall contained the highest concentration of label, with lesser amounts in the follicle cells and least in the oocytes. Oocytes and follicle cells localized at the periphery of the ovary were labeled first, and both cells became progressively labeled throughout the ovary with time; the label first appeared localized in the nucleolus of the oocyte.Sucrose gradient analysis of the separated cellular components of prelabeled hormone-treated ovaries indicated that RNA synthesis occurred in all segments of the ovary and that the spawned oocyte fraction was the least active. Synthesis of ribosomal RNA was detectable after a lag period of approximately 4 hr. Oocytes incubated in ³H-uridine during and subsequent to 1-methyladenine-induced spawning and maturation synthesized 15–19 S and low molecular weight RNA but not ribosomal RNA. Synthesis of the 15–19 S RNA was inhibited with ethidium bromide and to a limited extent by actinomycin D. Isolated mitochondrial fractions contained most of the labeled 15–19 S RNA. These data suggest the mitochondrial origin of most, if not all, of this intermediate-weight RNA. On the basis of these studies, it appears that starfish oocytes and follicle cells are metabolically active at the transitional period from growth to maturational stages in oocytes. Synthesis of RNA furthermore apparently continues in the cytoplasm subsequent to germinal vesicle breakdown and spawning.     

Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

To investigate the effect of recombinant bovine somatotrophin (rGH) on ovarian folliculogenesis in sheep, 18 mature Scottish Blackface ewes were assigned randomly to two treatment groups. Starting from day 5 of the synchronised oestrous cycle, animals were injected daily with either vehicle (control group) or 12.5 mg rGH (rGH-treated group) for 7 days. Blood samples were collected once daily during the experimental period for the measurement of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, follicle-stimulating hormone (FSH), luteinising hormone (LH) and progesterone. At the end of treatment animals were killed and ovaries collected. All follicles at least 1.0 mm in diameter were dissected out and diameters measured to assess follicular populations for individual animals. Five small follicles (1.0–3.4 mm in diameter) and all the large follicles (at least 3.5 mm) from each animal were incubated in 1 ml of Medium 199 for 1 h. Medium was then changed and incubation continued for a further hour. All medium samples were assayed for IGF-I, oestradiol, testosterone and progesterone.Treatment of ewes with rGH had no effect on the total number of follicles at least 1.0 mm in diameter (control, 34.4 ± 2.6; rGH-treated, 31.3 ± 1.4; P > 0.2). However, when follicles were further classified into different size categories (1.0–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, 5.1–6.0 and over 6.0 mm in diameter), the population of follicles 2.1–3.0 mm in diameter was significantly increased by rGH treatment (control, 9.2 ± 0.7; rGH-treated, 13.8 ± 1.1; P = 0.02). The number of follicles of 3.1–4.0 mm diameter in the rGH-treated group tended to be increased (P = 0.09), whilst the population of follicles 1.0–2.0 mm in diameter was reduced (P = 0.07). Treatment of ewes with rGH significantly increased peripheral concentrations of GH (P < 0.01), IGF-I (P < 0.01), insulin (P < 0.01) and progesterone (P < 0.05). There was no effect of rGH treatment on circulating concentrations of FSH and LH. Both large and small follicles from rGH-treated ewes secreted significantly (P < 0.001) more IGF-I (37.8 ± 2.2 ng ml h⁻¹, n = 50) than follicles from the control group (26.7 ± 1.6 ng ml⁻¹ h⁻¹, n = 73). However, there was no significant effect of rGH treatment on the secretion of oestradiol, testosterone and progesterone by either large or small follicles.It is concluded that treatment of mature ewes with rGH can enhance the development of ovarian follicles to the gonadotrophin-dependent stages. Furthermore, rGH appears to act through increased secretion of ovarian IGF-I, as well as increased peripheral concentrations of IGF-I and insulin.     

Follicular populations during the estrous cycle in heifers. II. Influence of right and left sides and intraovarian effect of the corpus luteum

Ovarian follicles ≥2 mm were studied in 22 Holstein heifers by daily ultrasound examinations. Data were partitioned by right vs. left ovary and corpus luteum bearing ovary vs. the contralateral ovary. There were significantly more (P < 0.03) follicles 4–6 mm, > 13mm and total ≥2 mm in the right ovary, regardless of the presence of a corpus luteum. Significantly more (P < 0.05) follicles 2–3 mm, > 13 mm and total ≥2 mm were observed in the ovary bearing the corpus luteum. Interactions between day and corpus luteum appeared to be due to a greater number of follicles in the ovary bearing the corpus luteum during the first part of the interovulatory interval. There was also a day by right side vs. left side interaction for the number of follicles > 13 mm. Interpretation of the interactions was that the presence of a corpus luteum was conducive to the development of more anovulatory diestrous follicles > 12 mm. However, as regression of the corpus luteum progressed, there was an apparent proclivity for preovulatory follicular development in the right ovary. There was no apparent pattern of alternating sides of ovulation or of alternating sides of development of anovulatory diestrous follicles and preovulatory follicles in heifers observed for more than one interovulatory interval. There was not a significant difference in the maximum diameter attained by the anovulatory diestrous follicle or preovulatory follicle between ovaries ipsilateral or contralateral to the corpus luteum; however, the maximum diameter attained by the preovulatory follicle was greater (P < 0.05) than that attained by the anovulatory diestrous follicle.     

Analytical and experimental studies on the relationship between Na+, K+, and water uptake during volume increases associated with Fundulus oocyte maturation in vitro

Summary Oocytes of marine and estuarine teleosts often undergo pronounced volume increases during the maturation phase of development that precedes ovulation and fertilization. To examine the physiological correlates of these volume increases, prematuration follicles of the saltmarsh teleost, Fundulus heteroclitus, were cultured in vitro with a maturation-inducing steroid (17-hydroxy-20-dihydroprogesterone). Mean follicle volume rose significantly (75%) during a 40-h incubation period. Similar to the situation previously found in vivo, uptake of water by the maturing follicle was responsible for this volume increase in vitro, with the water content increasing from 62% to 78% of the total follicle mass. The follicle contents of two probable osmotic effectors-Na⁺ and K⁺-also rose, the increase in K⁺ being twice that of Na⁺. The influx of K⁺ even exceeded water uptake, resulting in a net increase in the concentration of this cation. It thus appears that the influx of these cations, in particular K⁺, is a major cause of the uptake of osmotically obligated water and subsequent volume increase experienced by maturing F. heteroclitus follicles. In a search for operant mechanisms, it was found that follicle hydration, but not maturation, was strictly dependent on external K⁺ in a concentration-dependent manner. The mechanism by which K⁺ accumulates in the follicle was insensitive to ouabain, so that a typical Na⁺, K⁺-ATPase mechanism does not appear to be involved. The ability of external K⁺ to promote follicle hydration was gradually lost during the maturation process as the oocyte dissociated from the surrounding granulosa cells in preparation for ovulation. Removal of all associated somatic cells prior to maturation prevented subsequent steroid-initiated hydration but not maturation. The results suggest that K⁺ may be translocated from surrounding granulosa cells to the oocyte via gap junctions during maturation.Abbreviations GVBD germinal vesicle breakdown     

Developmental potential of LT/Sv parthenotes derived from oocytes matured in vivo and in vitro

Release of oocytes of LT/Sv mice from the meiosis-inhibiting influence of antral follicles promotes parthenogenetic activation and development to early cleavage stages of 14% of the eggs. However, to attain the potential to develop to blastocysts under the culture conditions used, the oocytes must mature within follicles for 8–9 hr after human chorionic gonadotropin (HCG) administration. The results suggest that some positive influence, which does not occur during spontaneous oocyte maturation under defined conditions in vitro, occurs within preovulatory follicles and imparts developmental competence to the maturing oocytes.     

<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis> gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays

Fractionation and characterization of gonadotropins (GtH) from Fundulus heteroclitus pituitary extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-exclusion chromatography eluted gonadotropic activity in one major protein peak (M_r ~ 30,000). Anion-exchange and hydrophobic-interaction chromatography (HIC) yielded two distinct peaks of 17beta-estradiol (E₂)- and 17alpha-hydroxy,20beta-dihydroprogesterone (DHP)-promoting activity with associated oocyte maturation. Two-dimensional chromatography (chromatofocusing followed by HIC) resolved pituitary extracts into two active fractions; both induced E₂ synthesis, but one was relatively poor in eliciting DHP and testosterone production. Thus, using homologous bioassays, at least two quantitatively different gonadotropic (steroidogenic) activities: an E₂-promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like), which has a lower isoelectric point but greater hydrophobicity than the former, can be distinguished from F. heteroclitus pituitaries by a variety of chromatographic procedures. This study complements previous biochemical and molecular data in F. heteroclitus and substantiates the duality of GtH function in a multiple-spawning teleost.     

Characterization of Luteinizing Hormone and Luteinizing Hormone Receptor and Their Indispensable Role in the Ovulatory Process of the Medaka

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare’s serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.     

Habitat use, movement, and growth of young-of-the-year Fundulus spp. in southern New Jersey salt marshes: Comparisons based on tag/recapture

We examined habitat use, movement, and growth of young-of-the-year (YOY) Fundulus heteroclitus and Fundulus luciae with a tag/recapture experiment in tide-dominated salt marshes to determine if movements from Spartina marsh surface can account for the occurrence of larger, older individuals in other habitats. Evaluation of the tagging techniques in laboratory experiments with YOY F. heteroclitus (15-35 mm TL) found that coded wire tags were retained at least up to 77 days. The high rates of recapture in the field also indicate that the tagging approach generally worked well. Of a total of 5748 YOY F. heteroclitus (14-40 mm TL) and 133 YOY F. luciae (17-40 mm TL) tagged, 56.0% and 74.4% were recaptured, respectively. Most (44%) YOY F. heteroclitus recaptured occurred at or near (0-5 m) the release site, but some were captured up to 299 m away up to 166 days after tagging. By comparison, movement of F. luciae was very limited, with 99% of recaptures occurring at the exact site of release after up to 66 days at liberty. These different movement patterns by YOY Fundulus indicate that species-specific behavior plays an important role in habitat selection. In addition, it appears that dispersal of YOY F. heteroclitus can help to explain the occurrence of larger individuals of this species in Phragmites-dominated marshes even though there is little evidence of use of this habitat by small YOY.     

Induction of cell DNA replication in G1-specific ts mutants by microinjection of SV40 DNA

Treatment of Xenopus laevis follicles with 50–100 units/ml of human chorionic gonadotropin causes rapid stimulation of [¹⁴C]glucose uptake. Studies with these follicles showed that the stimulation of uptake occurred with a wide range of concentrations of [¹⁴C]glucose or its nonmetabolizable analog [¹⁴C]3-O-methylglucose. Approx. 70% of the glucose taken up in both hormone-treated and control cells becomes incorporated into glycogen within 1 h. The uptake of sugar by these follicles was also stimulated by bovine-luteinizing hormone—but not by folliclestimulating hormone, progesterone or insulin. Human chorionic gonadotropin stimulated sugar uptake by follicles containing medium-sized oocytes (stages 3,4 and 5 according to Dumont) which cannot be induced to undergo meiotic maturation by this hormone. After 4–6 h treatment of fully grown X. laevis follicles with either progesterone or human chorionic gonadotropin, glucose uptake suffers a drastic decrease to below basal levels. This inhibition of uptake is coincident with the breakdown of the germinal vesicle of the oocyte and is clearly related to meiotic maturation, since it is not observed with medium-sized follicles which cannot mature.     

Loss of abnormal spindle-like,microcephaly-associated (Aspm) disrupts female folliculogenesis in mice during maturation and aging

The abnormal spindle-like, microcephaly-associated (ASPM) gene is a causative gene of autosomal recessive primary microcephaly (MCPH) 5 in humans, which is characterized by a reduction in brain volume. It was previously reported that truncated Aspm proteins in transgenic mice caused major defects in the germline, a severe reduction in ovary weight and the number of follicles accompanied by reduced fertility. However; it remains unknown whether a loss of Aspm induces abnormal ovarian function, resulting in female infertility. In order to assess the ovary function, we examined vaginal smear cytology from the age of 7 weeks to 100 weeks in CAG-mediated Cre-loxP conditional Aspm^-/- knockout mice and control female mice. In addition, we evaluated the ovarian size, fibrosis ratio and the number of follicles (primordial, primary, secondary, antral and atretic follicles) in mice from 15 weeks to 100 weeks old by image analyses. Mann-Whitney U-test was used for statistical analysis. The size of the ovary was significantly reduced in Aspm knockout mice at 15–20 weeks, 40–50 weeks and 70–80 weeks old compared with the control mice. Furthermore, at all stages, we found a severe decrease in the number of developing follicles at 10–15 weeks, 40–50 weeks and 70–80 weeks old, accompanied by disrupted cyclic changes of vaginal cytology and an aberrant upregulation of Foxo3, Kitl, and Lhcgr in Aspm knockout female. These results suggested that Aspm might play an important role in the folliculogenesis and estrous cyclicity of the postnatal ovary during maturation and aging.     

Quantification of Reproductive Isolating Barriers Between Two Naturally Hybridizing Killifish Species

Understanding the relative importance of various reproductive barriers to the early stages of speciation is an essential question in evolutionary biology. The closely related killifishes Fundulus heteroclitus and F. grandis occasionally hybridize in a small region in coastal Northeastern Florida showing that while barriers to reproduction exist, they are incomplete. The objective of this study was to elucidate barriers to reproduction between F. heteroclitus and F. grandis in the lab, as well as to quantify their strengths and relative contributions to reproductive isolation. Pre-zygotic (mating and fertilization) and post-zygotic (hatching) barriers were investigated by performing a variety of choice and no-choice laboratory mating experiments. Under no-choice conditions, barriers to mating had the greatest influence on hybrid production in F. grandis, whereas hatching barriers contributed to the majority of reproductive isolation in F. heteroclitus. Under choice conditions, however, pre-zygotic barriers had the greatest influence on hybrid production in both species. The total reproductive isolation that was observed in females of each species was stronger in F. heteroclitus than in F. grandis, and was nearly complete in F. heteroclitus females under choice conditions and was of moderate strength in F. grandis females. These results reveal an asymmetry in the potential gene flow between these two species, with F. grandis being more likely to hybridize than F. heteroclitus in the absence of environmental influences. No-choice backcrosses were also conducted and showed that at least some F1 hybrids are fertile. The observation that pre-zygotic barriers tend to be stronger than post-zygotic barriers in the early stages of speciation is consistent with similar studies in other organisms.     

Dietary energy source and feeding levels during the rearing period affect ovarian follicular development and oocyte maturation in gilts

Fifty-four Landrace × Yorkshire gilts (59.0 ± 4.2 kg and 147 ± 3 d old) were used to examine the effects of dietary energy source (starch or mixed fat) at high [112.5% of energy requirements recommended by NRC (1998)], normal (100%), and low (87.5%) energy feeding levels on ovarian follicular development and oocyte maturation. Forty-seven estrus gilts were slaughtered at Day 19 after the second estrus; oocytes were recovered from follicles >4 mm in diameter, and matured in vitro for 44 h. Gilts fed high-energy diets had more follicles >4 mm (mean, 25.8 vs. 19.1, P < 0.05) and more oocytes that reached metaphase II (80.3 vs. 64.0%, P < 0.05) than those fed the low-energy diet. Furthermore, gilts fed starch-rich diets had enhanced oocyte nuclear maturation relative to those fed fat-rich diets (75.4 vs. 68.0%, P < 0.05). Compared to the lower-energy feeding groups, high-energy feeding groups had higher (P < 0.05) blood concentrations of postprandial insulin (1562.4 vs. 990.0 ng/4 h), IGF-I (321.2 vs. 256.9 ng/mL), and LH pulses (2.7 vs. 1.4 pulses/6 h). Follicular fluid concentrations of IGF-I (198.5 vs. 143.1 ng/mL) and estradiol (152.6 vs. 124.8 ng/mL) were higher (P < 0.05) in the high-energy group than in the normal group. Compared with gilts fed the high-energy diet supplemented with fat, gilts fed the high-energy diet supplemented with starch had a tendency (P < 0.10) towards increased IGF-I concentration in both blood and follicular fluid, and improved oocyte nuclear maturation during culture in vitro. We inferred that starch-rich, high-energy diets during rearing may improve ovarian follicular development and oocyte maturation in replacement gilts.     

Site of production of meiosis-inducing substance in ovary of starfish

The site of production of meiosis-inducing substance (MIS), produced in the ovary under the influence of gonad-stimulating substance (GSS) taken from radial nerves, was studied with the starfish, Asterina pectinifera. The rate of oocyte maturation observed in isolated oocytes with follicles kept in sea water containing GSS (100 μg/ml) was generally low when a small number of eggs per unit quantity of sea water was used. However, with increased number of oocytes per milliliter of GSS-sea water, the maturation rate increased up to 100% (10⁴ eggs/ml). The supernatant of such an incubation mixture of oocytes and GSS contained an appreciable amount of MIS. When a large number of oocytes (10⁴/ml) was used, the rate of oocyte maturation increased with the rise in concentration of GSS. Isolated follicles were found to produce MIS when incubated in sea water containing GSS, suggesting that the site of production of MIS is the follicle cells. The physiological role of the follicle cells surrounding the full grown oocytes seems to be to produce MIS just before spawning under the influence of GSS.     

Evidence that Meishan and Large-White hybrid preovulatory follicles may differentially affect oocyte in vitro maturation and fertilization

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles ≥4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P<0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P<0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentrations of oestradiol and progesterone in the conditioned media did not differ between the breeds (P>0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.     

Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E₂), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E₂ + bFGF or FSH + LH + E₂ + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E₂ turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.     

Changes in size,hydration and low molecular weight osmotic effectors during meiotic maturation of Fundulus oocytes in vivo

• 1.1. Gains in the water content of the Fundulus heteroclitus follicle account for over 90% of the total wet weight increase, and consequentially most of the size increase, associated with meiotic maturation.
• 2.2. Increases in intracellular Na⁺ and K⁺ actually exceed the accompanying increases in oocyte water, resulting in net gains in the concentration of these solutes. Changes in oocyte osmolality during maturation are mostly closely paralleled by variations in the concentration of K⁺.
• 3.3. Concentrations of various free amino acids, including taurine, remain constant or decline during maturation.
• 4.4. Taken together, these results suggest that an influx of K⁺, followed by osmotically-obliged water, is a primary cause of water uptake during oocyte maturation in F. heteroclitus.

     

Gonadotropin-induced steroidogenic shift towards maturation-inducing hormone in Japanese yellowtail during final oocyte maturation

Intact ovarian follicles, obtained from untreated and human chorionic gonadotropin (HCG) treated Japanese yellowtail Seriola quinqueradiata during different maturational stages, were incubated with radioactive [3H]pregnenolone, [³H]17‐hydroxyprogesterone or [¹⁴C] androstenedione and steroid metabolites identified by thin layer chromatography (TLC) followed by recrystallization to constant specific activity. In untreated late vitellogenic (0 h) follicles, androstenedione was the major product with smaller amounts of testosterone and oestradiol‐17α. In post‐vitellogenic (12 h post‐injection) intact follicles, androstenedione predominated, and although testosterone and oestradiol‐17α were not produced, there were small amounts of 17, 20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) and 17,21‐dihydroxy‐4‐pregnene‐3, 20‐dione (11‐deoxycortisol). In HCG‐treated fish, a steroidogenic shift resulted in the disappearance of testosterone and oestradiol‐17 coinciding with the appearance of 17, 20β‐P. During early and late final oocyte maturation FOM (24 and 36 h post‐injection), there was a five‐ to seven‐fold increase in the production of 17, 20β‐P, whereas production of 11‐deoxycortisol remained almost the same. During FOM, in addition to 17,20β‐P, its 5β‐reduced metabolite, 17,20β‐dihydroxy‐5β‐pregnan‐3‐one (5β‐17,20β‐P) was synthesized, suggesting a decrease in maturation‐inducing 17,20β‐P activity. 17, 20β,21‐Trihydroxy‐4‐pregnen‐3‐one (20β‐S) was not synthesized by ovarian fragments in Japanese yellowtail at any maturational stage. The metabolites identified on TLC during FOM were tested to evaluate their maturation‐inducing activity in an in vitro bioassay. Of the steroids tested, 17,20β‐P was the most effective inducer of germinal vesicle breakdown (GVBD), followed by 5β‐17,20β‐P. Timely synthesis of 17,20β‐P immediately prior to and during FOM as well as its great potency in inducing GVBD in vitro supports the evidence for a physiological role of 17,20β‐P as a maturation‐inducing hormone in Japanese yellowtail.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.