In vitro effect of vitellogenin on steroid production by ovarian follicles of greenback flounder Rhombosolea tapirina

Ovarian follicles from vitellogenic greenback flounder (Rhombosolea tapirina) were incubated in L15 medium alone, or containing human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP) or the steroid precursors testosterone (T), 17-hydroxyprogesterone (17P) and androstenedione (A) in the presence of vitellogenin (Vtg) at 0.1-5.0mg mL(-)(1). Medium concentrations of 17beta-estradiol (E(2)) and T were measured by radioimmunoassay. HCG generally stimulated follicular E(2) but not T production, whereas 17P, A and T stimulated production of E(2), T, and E(2) respectively. Treatment of follicles with dbcAMP inhibited follicular E(2) production, but increased follicular T production at high doses. The effect of low concentrations of Vtg on follicular steroid production was variable; however, higher doses of Vtg significantly suppressed basal, hCG-, dbcAMP- and steroid precursor-stimulated follicular E(2) and T production. The results of this study show that high concentrations of Vtg may suppress follicular steroid production by interfering in the steroidogenic pathway. This suggests that Vtg may regulate its own production by limiting the ovarian production of E(2).     

Daily growth increments in the otoliths of juvenile greenback flounder, Rhombosolea tapirina Gunther, 1862

Otolith check marks of both tetracycline-treated and control juvenile greenback flounders, Rhombosolea tapirina, were attributed to the stress of transfer between aquarium tanks. Subsequent counts showed that otolith increments were deposited daily.     

Milt characteristics and plasma levels of gonadal steroids in greenback flounder Rhombosolea tapirina following treatment with exogenous hormones

Fish were treated with exogenous hormones, and milt and blood samples were collected for up to 96 h post‐treatment. Blood plasma samples were assayed for the gonadal steroids testosterone (T), 11‐ketotestosterone (11KT) and 17,20ß‐dihydroxy‐4‐pregnen‐3‐one (17.20ßP). Milt volume, spermatocrit and sperm motility were measured from milt samples. Non‐spermiated fish showed increased plasma T and 11KT in response to human chorionic gonadotropin (hCG) but not luteinising hormone releasing hormone analogue (LHRHa). Fish did not become spermiated in response to treatment with hCG, LHRHa, 11KT, 17‐hydroxyprogesterone (17P) or 17,20ßP. Spermiated fish showed an increase in milt volume in response to hCG and LHRHa but not exogenous steroids. Sperm motility declined to zero over 120 s and was not affected by hormone treatment or sampling time. Increased milt volume was accompanied by increased plasma T and 11KT, but not 17.20ßP levels. In a separate experiment, LHRHa delivered by injection or pellet was equally effective at increasing milt volume but had no effect on plasma steroid levels. Spermatocrit declined with stripping but was not affected by hormone treatment, nor was sperm motility. Co‐treatment of fish with 17P plus LHRHa had no additive effect on plasma steroid concentrations or milt volume. The results suggest that as in other teleosts, gonadotropin mediates milt production in greenback flounder.     

Relationships between protein-nitrogen flux and feeding regime in greenback flounder, Rhombosolea tapirina (Günther).

This study aimed to measure protein synthesis and growth performance in flatfish, Rhombosolea tapirina, fed on three feeding regimes: 1% body wt. fed in the morning (MM1); 2% body wt. fed in the morning (MM2) and 2% body wt. fed in equal amounts in the morning and in the afternoon (MA2). After 6 weeks there were no significant differences in wet wt., protein growth or efficiency of protein-nitrogen flux. Significantly lower protein consumption on MM1 resulted in significantly lower rates of protein synthesis and degradation compared with MM2 but not MA2. After 18 weeks there were still no significant differences between feeding regimes although trends in growth and efficiency reflected the situation after 6 weeks. Fish were maintained individually, and when analysed on this basis, there were significant relationships between protein consumption and protein synthesis [k(s)=0.92, k(r)+1.16 (n=17; r(2)=0.49; P<0.005)], between protein consumption and protein degradation [k(d)=0.82, k(r)+1.06 (n=17; r(2)=0.41; P<0.005)] and between average daily protein consumption and protein growth [k(g)=0.15, k(rx)-0.15 (n=17; r(2)=0.75; P<0.001)]. Thus, increasing protein consumption resulted in increased protein synthesis and growth. However, the combination of high rates of protein synthesis and protein degradation in relation to protein consumption, and resulting high anabolic stimulation and low retention of synthesised protein, contributed to the low growth performance of greenback flounder.     

In vitro culture of mouse GV oocytes and preantral follicles isolated from ovarian tissues cryopreserved by vitrification

Cryopreservation of ovarian tissues containing many immature oocytes occurs in both gamete/embryo research and clinical medicine. Using vitrification, we studied factors related to meiosis after cryopreservation using the COCs (cumulus oocyte complexes) and preantral follicles obtained from cryopreserved ovarian tissues. COCs were isolated and cultured for 17 approximately 19 hr. Thereafter, Metaphase II stage (MII stage) oocytes and fertilized oocytes after IVF were observed at a rate of 76.5% and 60.0%, respectively. Preantral follicles (100 approximately 130 microm in diameter) were isolated and cultured in alpha MEM containing hFSH, ITS, and FBS. HCG and EGF were added to the media to stimulate ovulation on the 12th day of culture. The survival rates of the follicles obtained from the frozen/thawed ovaries were 66.4%. After 12 days of culture, the diameter of the follicles isolated from fresh (620.2 +/- 11.3 microm) and frozen/thawed ovaries (518.7 +/- 15.1 microm) differed as did the estradiol concentrations (3474.2 +/- 159 pg/ml vs. 1508.2 +/- 134 pg/ml). After in vitro ovulation, MII stage oocytes were observed in 84.5% of the fresh group and 60.5% of the frozen/thawed group while the fertilization rate was 74.2% and 53.5%, respectively. These studies demonstrate that cryopreservation of mouse ovarian tissues by vitrification did not affect the oocyte's ability to undergo meiosis. Thus, this technique may become a powerful tool for the preservation of the female gamete.     

Recent achievements in in vitro culture and preservation of ovarian follicles in mammals

The mammalian ovary contains a large number of follicles that are in various developmental stages. The largest portion of them are primordial follicles. However, throughout the female reproductive lifespan only a small proportion of these follicles will produce oocytes competent to undergo successful maturation and ovulation. The rest of the ovarian oocytes (>99.9%) undergo atresia. It would be of great practical benefit to rescue some of these follicles by growing them in culture in order to provide an extra source of gametes. There is considerable interest in developing technologies that aim to produce fully-grown, developmentally competent oocytes from a pool of early developmental stages of follicles. Two methods have been used: 1/ long-term in vitro culture of either follicles or oocytes, and 2/ transplantation of ovarian tissue grafts. The development of efficient technologies may provide an additional source of oocytes for livestock production and reproduction in humans and rare or endangered species. The aim of this paper is to present a comprehensive review of recent achievements in the utilization of small ovarian follicles (primordial, preantral and early antral) by long-term in vitro culture and/or transplantation of ovarian tissue grafts (fresh and cryopreserved) in mammals including humans.     

Comparison of three in vitro culture systems for maturation of early preantral mouse ovarian follicles

The aim of this study was to compare three different culture systems for in vitro follicular growth and oocyte maturation in ovarian follicles of mice in order to assess the technique with the optimal growth and improved rate of meiotic maturation. The three systems tested were culture under oil, on a hydrophobic membrane and on agar respectively. Early preantral follicles were cultured for 12 days in alpha-MEM GlutaMAX medium. Follicular growth, oocyte meiotic maturation, oocyte extrusion, atresia and estradiol production were analysed. Follicular development showed two phases in the three systems, with slow growth before day 5 and subsequent acceleration. The percentage of follicles transferred into oocyte maturation medium was significantly higher after culture under oil. The proportion of oocytes that achieved nuclear maturation (metaphase II) was higher when follicles were cultured under oil or on a hydrophobic membrane than on agar. Our results support the use of culture under oil for in vitro follicular growth from the early preantral stage in order to obtain metaphase II oocytes. Fertilization ability of these oocytes and the capacity to obtain healthy mice in a reproducible manner warrants further investigation.     

In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue

In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.     

In vitro steroid production by isolated ovarian follicles of the striped trumpeter

Ovarian follicles from striped trumpeter Latris lineata were incubated in L15 medium alone, or medium supplemented with gonadotropin (GtH) preparations (human chorionic GtH, carp maturational GtH or partially purified salmon GtH), testosterone (T) or 17-hydroxyprogesterone (17P). Levels of oestrone (E₁), 17 β -oestradiol (E₂), T, and 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β P) in the medium after incubation were measured by radioimmunoassay. Basal production of E₂ was high from previtellogenic follicles, whereas little T was produced. Both T and E₂ production increased in response to treatment with GtH or steroid precursors. Vitellogenic follicles showed basal production of both T and E₂, and T but not E₂ levels generally increased in response to hormone treatment. Preparations containing follicles nearing final maturation showed low basal production of E₂ but high production of T. Treatment with steroids resulted in little change in E₂ but often very large increases in T production, whereas GtH stimulated lesser increases. 17,20 β P production was detectable from incubations of maturing follicles from two out of five fish, and in those two incubations, increased in response to treatment with 17P. E₁ was not detectable in any incubations. The results indicate that there is a shift in steroidogenesis from E₂ to T production during oocyte development, and provide further evidence that steroid biosynthesis in non-salmonids is principally regulated by substrate availability.     

Characterization of in vitro growth of bovine preantral ovarian follicles: A preliminary study

Preantral follicles were mechanically extracted from bovine ovaries collected at slaughter. On average 90 early growing follicles were collected per ovary. The follicles were cultured for 7 days in Dulbecco's modified Eagle medium F-12 nutrient mixture + 10% fetal calf serum + 10% newborn calf serum. The individual follicular diameter and the follicular DNA content were recorded at the start and the end of culture. The follicular DNA content was estimated by a microfluorometric method using the fluorochrome, 4'-6 diamidino-2-phenylindol-2HCl (DAPI). After culture, 86% of the follicles looked morphologically normal. Of the surviving follicles, 73% showed an increase in diameter varying between 5 and 30 mum, and the follicular DNA content increased from 1 to 72% under the same culture conditions. These results indicate that bovine preantral follicles can survive in vitro and even grow in a single medium.     

In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue

Achieving full in vitro growth of oocytes of both domestic animals and humans remains a major challenge. The objective of this study was to examine the in vitro development of primary follicles isolated enzymatically from cryopreserved sheep ovarian tissue. In Experiment 1, isolated primary follicles (mean diameter 60.1+/-0.78microm) were cultured in serum-free medium on fibronectin-coated wells for 42 days. Initially follicular structure was lost as granulosa cells plated down, but by Day 7 two distinct morphologies began to emerge. Nineteen out of 36 oocytes were gradually re-surrounded by granulosa cells, forming follicle-like units (reorganized follicles), and the remaining 17 were not (non-reorganized follicles). On Day 2, there was no difference in diameter of oocytes between reorganized and non-reorganized follicles. The diameter (mean+/-S.E.M.) of oocytes of reorganized follicles increased (P<0.05) from 47.1+/-2.2microm to 65.3+/-2.6microm between Day 2 and Day 42, respectively, but that of oocytes of non-reorganized follicles showed no change. In Experiment 2, oocyte growth and granulosa cell differentiation during long-term culture of primary follicles (>42 days) were examined. Oocytes of reorganized follicles reached a maximum diameter of 75.4+/-2.0microm, a size equivalent to that of oocytes of ovine secondary follicles. Using RT-PCR, mRNA for follicle stimulating hormone receptor was detected in granulosa cells of freshly isolated secondary follicles and of long-term cultured reorganized follicles, but not of non-reorganized follicles. In Experiment 3, we tested if the culture conditions could support further oocyte growth in secondary follicles. The oocytes from enzymatically isolated secondary follicles increased in diameter from 77.7+/-1.6microm to 98.8+/-2.1microm (P<0.05) during 28 days in culture. The changes in oocyte size and in gene expression by granulosa cells support the conclusion that isolated ovine primary follicles developed in vitro to reach the secondary follicle stage.     

人腔前卵泡分离及培养

采用机械吹打、胶原酶消化、镜下显微解剖及胶原酶消化加镜下显微解剖4种卵泡提取方式并相互对比.将分离得到的腔前卵泡在含FSH0.5IU/mL、1.0IU/mL和2.0IU/mL的培养液中培养,检测其E2分泌量.与机械吹打、胶原酶消化相比,胶原酶消化加镜下显微解剖法不仅提取卵泡多(P＜0.01),而且可以得到原始、初级、次级各级卵泡.但操作时间较长(P＜0.01).得到的腔前卵泡在FSH为0.5IU/mL、1.0IU/mL和2.01IU/mL的培养液中培养,所分泌的E2分别为10.86pg±4.11pg、31.55pg±9.34pg和43.82pg±18.76pg,较之对照组的4.99pg±2.09pg有显著性差异(P＜0.01),E2的分泌量与FSH浓度呈剂量依赖性关系.FSH有促进腔前卵泡分泌E2的作用.     

Mouse ovarian follicles secrete factors affecting the growth and development of like-sized ovarian follicles in vitro

A series of experiments have been carried out to determine whether follicles secrete factors able to affect the growth and development of other, like-sized follicles. Late preantral mouse ovarian follicles were either cocultured or cultured in media conditioned by previously cultured follicles. In particular, the experiments examined whether follicles do secrete such factors, whether the level of FSH in the culture media can affect that process, and what the nature of such secretory factor(s) might be. First, pairs of follicles were cocultured across a polycarbonate membrane containing pores. This showed that communication between the follicles resulted in the stimulation of growth and that the stimulation was due, at least in part, to the production of secretory factor(s). In subsequent experiments, follicles were cultured in media that had been preconditioned by previously cultured follicles. The concentration of FSH in the cultures determined the effect of the conditioned media: conditioned media was stimulatory to follicle growth when levels of FSH remained high throughout the culture, but inhibitory when FSH levels were dropped midway through the cultures. Heat inactivation removed this inhibitory effect, showing that the factor was likely to be a protein; addition of follistatin to the conditioned media did not alter its effect, indicating that the factor was unlikely to be activin. We have shown through a series of culture experiments that mouse follicles secrete factor(s) that can affect the development of other like-sized follicles when cultured from the late preantral to Graafian stages. Furthermore, we have shown that the effect (or production) of such factors is dependent on the FSH environment of the follicles.     

In vitro culture of bovine preantral follicles

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture.The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them.From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.     

Regeneration of atretic sheep ovarian follicles in vitro.

Large (4--6 mm diam.) and small (2--3 mm) atretic follicles were removed from sheep ovaries during the luteal phase of the cycle and maintained in organ culture without hormonal supplementation for up to 5 days. The structure, cell dynamics and steroid-producing capacity of the follicles were compared with those of non-atretic follicles of similar size. The granulosa layer of the atretic follicles invariably regenerated in culture, increasing in thickness more than 2- and 4-fold in large and small follicles respectively. This could not be accounted for by cell division which remained low throughout the culture period. In contrast, non-atretic follicles showed high mitotic activity during the first 24 h in culture: this was not associated with an increase in granulosa thickness in large follicles although there was a 4-fold increase in small ones. An increase in internuclear spacing, a measure of cell size plus intercellular space, partly accounted for the increase in granulosa thickness in atretic follicles. Even when granulosa cells remained in close apposition there was an almost total absence of gap junctions, a prominent feature in the granulosa of non-atretic follicles both in vivo and in vitro. Pyknotic nuclei and atretic bodies rapidly disappeared from the regenerating granulosa layer. The theca interna was restored in culture to a state ultrastructurally closely resembling that of non-atretic follicles in vivo. Total steroid secretion (oestradiol-17beta, testosterone plus progesterone) into the culture medium (pmol.mg tissue-1.24 h-1) was the same for atretic and non-atretic follicles of comparable size. There was, however, a marked difference in the type of steroid produced, largely related to a loss of aromatizing capacity in atretic follicles. The predominant steroid secreted by large non-atretic follicles was oestrogen, with slightly smaller amounts of testosterone, whereas the principal steroid secreted by large atretic follicles was progesterone. In small non-atretic and atretic follicles, the predominant steroid was testosterone, but the non-atretic follicles also secreted appreciable amounts of oestrogen. Addition of FSH to the culture medium did not restore aromatizing capacity to the atretic follicles.     

In vitro development of caprine ovarian preantral follicles

The in vitro growth and developmental pattern of caprine preantral follicles cultured in agar gel was observed. Preantral follicles 50 to 150 microm in diameter were isolated from prepuberal goat ovaries by treatment with collagenase and DNase. The isolated preantral follicles were cultured in agar gel for up to 14 days. A group of 10 follicles in different developmental stages was cultured in a culture well coated with 0.6% agar gel and filled with DMEM medium supplemented with FCS (10%), hypoxanthine (2 mmol/mL), dbcAMP (2 mmol/mL), FSH (100 ng/mL), insulin-transferrin-selenium (ITS) (50 ng/mL), IGF-1 (50 ng/mL), hydrocortisone (40 ng/mL) and antibiotics. Follicle viability was determined under an inverted phase-contrast microscope according to morphological and histological criteria, and follicle growth was assessed by their size and appearance. The results showed that the three-dimensional structures and forms of follicles were basically maintained intact during culture. Primary follicles developed into secondary follicles and a few of them into antral follicles. A large portion of secondary follicles entered the antral stage, and oocytes also acquired growth. The formation of theca lamina and zona pellucida was observed. The survival capacity of secondary follicles was greater than primary follicles. The survival rates for primary and secondary follicles were 11.36% (5/44) and 71.16% (53/74), respectively. During in vitro development the follicles demonstrated dominance. This experiment revealed the preliminary characteristics of the in vitro development of caprine preantral follicles.     

Development of in vitro culture method for early stage zebrafish (Danio rerio) ovarian follicles for use in cryopreservation studies

There have been no reported methods for in vitro growth of early stage ovarian follicles for fish and their cryopreservation is still under investigation. If cryopreservation of early stage ovarian follicles can be achieved, in vitro procedures for ovarian follicle culture, development, ovulation and fertilisation after cryopreservation would be needed. The aim of the present study was to develop an in vitro culture method for early stage zebrafish ovarian follicles for use after their cryopreservation. Procedures for in vitro culture of stage I (primary growth) and stage II (cortical alveolus) ovarian follicles were developed. The effects of concentration of L-15 medium, pH and the concentration of human chorionic gonadotropin (hCG) and activin A were studied. The results demonstrated that early stage zebrafish ovarian follicles can be cultured in vitro for 24 h, stage I and II ovarian follicles can grow to the sizes of early stage II and stage III ovarian follicles after hCG treatment. The method developed here is effective for assessing early stage zebrafish ovarian follicles growth competence in vitro. The results from the present study indicated that in vitro culture is the most reliable method for assessing ovarian follicle viability when compared with vital dye staining methods.     

Influences of FSH and EGF on primordial follicles during in vitro culture of caprine ovarian cortical tissue

Factors that control the onset of folliculogenesis are critical to female gamete production, but poorly understood. The aim of the present study was to investigate the effects of FSH and EGF on the activation and growth of goat primordial follicles in vitro. To this end, pieces of goat ovarian cortex were cultured in vitro for 1, 3 or 5 days, at 39 degrees C in an atmosphere containing 5% CO(2), in minimum essential medium supplemented with insulin, transferrin, selenium, pyruvate, glutamine, hypoxanthine, BSA, penicillin, streptomycin and fungizone and with or without FSH (100 ng/ml) and/or EGF (100 ng/ml). At the end of the culture periods, the relative proportions of primordial, intermediate, primary and secondary follicles were calculated and compared with those in non-cultured tissue. In addition, mitotic activity of granulosa cells was studied by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In brief, it was found that goat primordial follicles activate spontaneously during culture in vitro and, while neither FSH nor EGF affected the proportion of primordial follicles that entered the growth phase, both stimulated an increase in oocyte and follicle diameter, especially in intermediate and primary follicles cultured for 5 days. On the other hand, there was no significant effect of culture or either growth factor on the proportion of PCNA-stained growing follicles. Contrary to expectations, neither FSH nor EGF affected follicle viability or integrity during culture, since the percentages of intact follicles did not differ between control, FSH and/or EGF containing medium. In conclusion, this study demonstrated that goat primordial follicles activate spontaneously in vitro, and that both FSH and EGF stimulate an increase in follicle size by promoting oocyte growth.