Ovarian Atresia Research Articles

Induced spawning and early ontogeny of New Zealand freshwater eels {Anguilla dieffenbachii and A. australis)

Knowledge on the reproductive biology of New Zealand freshwater eels (Anguilla australis Richardson and A. dieffenbachii Gray) is limited to changes associated with gametogenesis in the adults; details pertaining to embryonic and larval development are not available, but are of obvious benefit for a better understanding of the eel life history and for artificial propagation programmes, whether for aquaculture or conservation purposes. Therefore, eels were artificially matured and eggs fertilised with sperm from either species to investigate early ontogeny. Only eggs from A. australis hatched successfully after c. 45 h of incubation at room temperature, yielding larvae of c. 2.5 mm in length. Larvae survived for up to 5 days, by which time they had attained a length of 5.3 mm. Scanning electron microscopy of 1‐ and 2‐day‐old larvae revealed the presence of prominent free neuromasts on the head and along the flanks. Although hatching of New Zealand eels was achieved for the first time, it is strongly recommended that hormone treatment be initiated immediately following capture, well before ovarian atresia starts.

Species-specific Differences in Apoptotic Cell Localization in Granulosa and Theca Interna Cells during Follicular Atresia in Porcine and Bovine Ovaries.

Species-specific differences in the process of apoptosis in granulosa and theca layers during follicular atresia in porcine and bovine ovaries were investigated by in situ DNA 3’end-labeling (TUNEL) at the level of individual cells. In porcine ovaries, granulosa cells located on the inner surface of the granulosa layer appeared to undergo apoptosis at the first stage, followed by neighboring granulosa cells. In the contrast, granulosa cells located on the middle region appeared to undergo apoptosis at the first stage in bovine ovaries. In both porcine and bovine ovaries, detachment and degeneration of the granulosa cell layer and fragmentation of basement membrane occurred in the follicles at advanced stage of atresia. In the ovaries of both species, theca interna cells were still intact at the stage of early atresia and then apoptotic cells appeared in the late to final stages of atresia, but no apoptotic cells were observed in theca externa layers during follicular atresia. The present findings indicated that apoptosis occurring in granulosa cells is an initial symptom of follicular atresia in the ovaries of both species, and that the apoptosis inducing factor(s) and/or survival factor(s) for the granulosa cells may be different between porcine and bovine ovaries.

Bcl-X(LONG) protein expression and phosphorylation in granulosa cells.

Expression of Bcl-x protein was evaluated in hen ovarian follicles, relative to stage of development, and in cultured granulosa cells after treatment with various apoptosis-suppressing or -inducing agents. Using Western blot analysis, Bcl-X(LONG) was most frequently observed migrating as a doublet with a molecular mass of approximately 28 kDa; the apparent higher molecular mass band of this doublet was determined to represent a phosphorylated form. Consistent with previous findings reported for bcl-x messenger RNA, only the Bcl-X(LONG) (apoptosis-suppressing) form of protein was detected in the hen granulosa cells, and highest levels of Bcl-X(LONG) protein (sum of the protein doublet) expression were found in granulosa from preovulatory follicles together with tissues with immune function (e.g. spleen and bone marrow). Evidence for Bcl-X(SHORT) expression was found only in the theca and several nonovarian tissues. Immunocytochemical analysis of preovulatory vs. prehierarchal follicles confirmed the comparatively greater expression of cytoplasmic Bcl-X(LONG), particularly in preovulatory follicle granulosa. Levels of Bcl-X(LONG) were significantly increased after 20 h of culture in the presence of 8-bromo-cAMP (8-br-cAMP; compared with culture in control medium) in granulosa cells from both stages of follicle development. Such results are correlated with the protein's proposed function to protect against cell death in apoptosis-resistant, preovulatory follicle granulosa cells and are consistent with the ability of this cAMP agonist to increase bcl-X(LONG) messenger RNA levels in cultured cells. Furthermore, several factors that have previously been demonstrated to suppress apoptosis in granulosa cells, in vitro, (e.g. 8-br-cAMP, LH, FSH) were found to rapidly (within 15 min) increase levels of phosphorylated Bcl-X(LONG), compared with control cells, whereas an inhibitor of protein kinase A (H-89) blocked such phosphorylation. By comparison, transforming growth factor alpha, a factor previously found to attenuate apoptosis and apoptosis-inducing agents (e.g. paclitaxel, C8-ceramide, daunorubicin, UV irradiation) failed to phosphorylate Bcl-X(LONG). From these studies, it is concluded that both the phosphorylation of Bcl-X(LONG) (a short-term response) and increased levels of Bcl-X(LONG) (a comparatively slower response) in hen granulosa cells are promoted by gonadotropins via the adenylyl cyclase/cAMP signaling pathway. Moreover, elevated levels of chicken Bcl-X(LONG) protein expression and its phosphorylated state are correlated with resistance to apoptotic cell death in granulosa cells in vitro and ultimately a resistance to ovarian follicle atresia in vivo.

Boo, a novel negative regulator of cell death, interacts with Apaf-1.

In this report, we describe the cloning and characterization of Boo, a novel anti-apoptotic member of the Bcl-2 family. The expression of Boo was highly restricted to the ovary and epididymis implicating it in the control of ovarian atresia and sperm maturation. Boo contains the conserved BH1 and BH2 domains, but lacks the BH3 motif. Like Bcl-2, Boo possesses a hydrophobic C-terminus and localizes to intracellular membranes. Boo also has an N-terminal region with strong homology to the BH4 domain found to be important for the function of some anti-apoptotic Bcl-2 homologues. Chromosomal localization analysis assigned Boo to murine chromosome 9 at band d9. Boo inhibits apoptosis, homodimerizes or heterodimerizes with some death-promoting and -suppressing Bcl-2 family members. More importantly, Boo interacts with Apaf-1 and forms a multimeric protein complex with Apaf-1 and caspase-9. Bak and Bik, two pro-apoptotic homologues disrupt the association of Boo and Apaf-1. Furthermore, Boo binds to three distinct regions of Apaf-1. These results demonstrate the evolutionarily conserved nature of the mechanisms of apoptosis. Like Ced-9, the mammalian homologues Boo and Bcl-xL interact with the human counterpart of Ced-4, Apaf-1, and thereby regulate apoptosis.

Spawning frequency, fecundity, egg size and ovarian histology in groups of Tilapia zillii maintained upon two distinct food ration sizes from first-feeding to sexual maturity

Duplicate groups of first-feeding (4-5-d-old) Tilapia zillii (Gervais) were maintained on two feeding regimes (high and low ration size) for a total period of 550 d (~ 17 months) in environmentally-controlled aquaria. Differences in mean individual fish weight were evident between fish fed the different ration sizes after just 3 months, though these differences were not statistically significant until day 180. Thereafter, fish maintained upon the higher ration were significantly larger than fish fed a reduced ration. Mortality in high ration fish over the course of the experiment was ~60 % compared to ~88 % in low ration fish. Mortality in both treatment groups was most pronounced during the first three months (particularly during the first 30 d) but was much reduced there-after. Spawning trials undertaken from day 450 onwards indicated that there was no significant difference between the two ration groups in terms of spawning periodicity over a discrete time period of 60 d. Mean total fecundity was significantly higher in fish on high rations though mean egg diameter and gonadosomatic index remained unchanged. Adjustment of spawning data to a common maternal fish size using one-factor ANCOVA failed to detect significant differences between the two ration levels in terms of either mean total fecundity or mean egg diameter. Stereological analysis of plastic-embedded ovarian tissue revealed that the ovaries of low ration fish possessed significantly more stage 2 and 3 (early and late perinucleolar) oocytes and significantly less stage 6/7 (late-vitellogenic/maturing) oocytes than the ovaries of fish fed the higher ration. No significant differences were detected between the two rations in terms of ovarian atresia. It is suggested that under prolonged food restriction, female T. zillii sacrifice somatic growth such that reproductive investment can be maintained.

Ovarian atresia in cephalopods

Ovarian atresia is described for the first time in cephalopods. Detailed histological examination of ovaries of Loligo vulgaris reynaudii in various stages of maturity revealed atresia in primary, previtellogenic and vitellogenic oocytes. These were analysed separately. Previtellogenic and vitellogenic oocytes could be subdivided into five clear stages, whereas primary atretic oocytes are given a general description. Follicular atresia lowers the number of maturing and mature eggs in the ovary, so previous fecundity estimates for cephalopods may have been too high. To correct potential fecundity estimates for losses arising from atresia, it is necessary to estimate the duration of each atresia stage in relation to the total spawning period.

Intracellular mechanisms of ovarian cell apoptosis

Apoptosis is an active cell `suicide' essential for the elimination of superfluous cells during diverse physiological processes in essentially all animal species. Although regulation of apoptosis by extracellular mediators is cell type-specific, new insights based on characterization of conserved intracellular effectors have suggested that intracellular pathways leading to apoptosis in diverse organisms is regulated by a group of evolutionarily conserved genes including ced-9/Bcl-2, ced-4/Apaf-1 and ced3/caspases gene families. To study whether the Bcl-2 family proteins are important in the regulation of ovarian cell apoptosis, we have used transgenic mice and yeast 2-hybrid protein–protein interaction assay to characterize the roles of Bcl-2 family proteins in ovarian atresia. The use of 2-hybrid analysis resulted in the isolation of a novel pro-apoptotic Bcl-2 protein, Bcl-2-related ovarian killer (Bok) and the identification of upstream mediators for ovarian cell apoptosis.

Evidence for the differential regulation of ovarian follicle responsiveness to human chorionic gonadotropin in vitro in a serranid teleost, Centropristis striata

Pre-maturational or full-grown ovarian follicles were isolated from captive, spawning black sea bass, Centropristis striata, at different times during the spawning season and tested for their ability to undergo oocyte maturation in vitro in response to human chorionic gonadotropin (hCG). The gonadosomatic index (GSI) was greatest in females collected during the mid-spawning period and the number of abnormal (atretic) follicles in the ovary progressively increased throughout the spawning season. The maturational response of follicles induced by hCG generally declined as the spawning interval progressed; an increased inhibition of oocyte maturation by doses ≥ 50 IU ml −1 hCG was also observed. Linear regression analyses indicated that the percentage of inhibition of oocyte maturation by 50, 100 and 500 IU ml −1 hCG was positively correlated with increasing levels of atresia within the ovaries from which the follicles were isolated. In females with low incidence of ovarian atresia (0–5%), follicular maturation achieved with 1–10 IU ml −1 hCG positively correlated with the GSI. However, when follicles isolated from females with a GSI ≥ 8 were treated with 50–500 IU ml −1 hCG the maturational response of follicles was reduced in comparison with the maturation of follicles from females with lower GSI that were treated with the same doses of hCG. In addition, doses of 100 and 500 IU ml −1 hCG appeared to be inhibitory on oocyte maturation once the GSI ≥ 8. These results suggest that the sensitivity of C. striata pre-maturational follicles to gonadotropin (GtH) may be regulated differently depending on both the overall state of follicular development and the levels of atresia within the ovary; the desensitization of GtH receptors may be one of the mechanisms possibly involved. It is recommended that the specific physiological condition of the ovary must be considered in selecting the timing and the dose of hCG to induce ovulation in this species.

Expression of Fas-Fas ligand system associated with atresia in murine ovary.

We detected that Fas receptor (Fas) was expressed at transcriptional levels in oocytes/eggs and some granulosa cells of murine ovary, whereas, Fas ligand (FasL) was found to be strongly expressed in granulosa cells by means of in situ hybridisation. These results were supported by an indirect immunofluorescence (IIF) test with anti-Fas monoclonal antibody (mAb)/FasL Ab. The lysates from granulosa cells were precipitated by anti-FasL Ab, exhibiting a specific band at 40 kDa. When zona pellucida (ZP)-free eggs were incubated with granulosa cells in vitro, apoptosis (DNA fragmentation) was induced in the eggs, as detected by the terminal deoxynucleotide transferase mediated dUTP-nick end labelling (TUNEL) method. To examine whether FasL-bearing cells can induce apoptosis in eggs expressing Fas through molecular interaction between FasL and Fas, we employed a baculovirus expression system to generate FasL on the surface of Spodoptera frugiperda (Sf9) cells. The co-incubation of eggs with Autographa californica nuclear polyhedrosis virus (AcNPV) and FasL transfected Sf9 (Sf9-FasL) cells in vitro was performed and resulted in the induction of apoptosis in eggs as detected by the TUNEL method. Apoptosis was absent in eggs co-incubated with AcNPV-1393 transfected Sf9 (Sf9-1393) cells. Thus, ovarian atresia was suggested to be induced by molecular interaction between FasL on granulosa cells and Fas on oocytes/eggs during oogenesis.

Expression and role of Fas-Fas ligand system in murine ovarian atresia

Expression and role of Fas-Fas ligand system in murine ovarian atresia

Early onset of deoxyribonucleic acid fragmentation during atresia of preovulatory ovarian follicles in rats.

Atretic demise rather than ovulation is the ultimate fate of the vast majority of ovarian follicles in mammals, affecting 70-99.9% of the follicles in various species. Recent studies have established that atretic degeneration of follicles is an apoptotic process, heralded by endonuclease degradation of DNA at internucleosomal sites, which generates DNA fragments in size multiples of 185-200 bp that are seen as distinct ladder bands after agarose gel electrophoresis. Using the well-characterized model of inducing atresia of preovulatory follicles in vivo by hypophysectomy and analyzing DNA fragmentation by autoradiography of size-fractionated DNA labeled at the 3' ends by [12P] dideoxy-ATP, we have examined the timing of atretic changes. DNA degradation was related to morphological signs of atresia, ovulability, and changes in follicular steroidogenesis. Rats were hypophysectomized on the morning of the day of proestrus, after which largest follicles were collected at various times. DNA fragmentation was analyzed in groups of five follicles. The increase in DNA fragments of low molecular weight up to 4 h after hypophysectomy was negligible (101 +/- 10%; 0 h time = 100%) but progressed 8, 12, 24, 48, and 72 h after hypophysectomy (143 +/- 20%, 168 +/- 27%, 235 +/- 29%, 3299 +/- 1075%, and 2249 +/- 805%, respectively; p < 0.03, n = 5). At 48 and 72 h, the extent of DNA degradation was higher than that observed in follicles cultured in a serum-free medium for 24 h. Likewise, staining of DNA by 4',6-diamido-2-phenylindole hydrochloride revealed apoptotic nuclei at 8 h after hypophysectomy (p < 0.01), and the percentage of such nuclei progressively increased afterwards. Thus, the increase in DNA fragmentation appeared concomitantly with atretic changes observed in previous studies (a decrease in ovulability at 6 h, and a spontaneous increase in progesterone accumulation and decrease in androgen and estrogen in follicles explanted 6 h after hypophysectomy) and preceded atresia detectable by morphological changes at 24 h. Detection of internucleosomal DNA degradation in preovulatory follicles early in the atretic cascade underscores the central role of apoptosis in ovarian follicle atresia.

Spawning frequency of the anchovyEngraulis capensis

Spawning frequency was determined for the first time for the South African anchovy Engraulis capensis using the incidence of females with one-day-old post-ovulatory follicles. Daily spawning frequency attained values of 15, 12, 10, 9,12 and 11% during November of 1985, 1986, 1987, 1988, 1989 and 1990 respectively. At these frequencies, each female anchovy on average spawned a new batch of eggs every 7–11 days. The proportion of the population spawning each day fell to 7% in February/March 1987, later in the spawning season, indicating that the mean interval between spawning was 14 days at mat time. Daily spawning activity was mostly around 22:00, based on the incidence of anchovy with both hydrated oocytes and post-ovulatory follicles less than 24 h old. A high incidence of atresia in ovaries was found in November 1988, probably as a result of poor availability of food.

Indicators of reproductive development in prespawning female winter flounder (Pleuronectes americanus) from urban and non-urban estuaries in the northeast United States

The relationship between exposure to xenobiotic compounds and ovarian development was evaluated in prespawning female winter flounder (Pleuronectes americanus) sampled from eleven sites on the northeast coast of the United States during the 1988 and 1989 spawning seasons. Three sites were located in Boston Harbor, MA, four sites were in Raritan Bay, NJ and four sites were in nearby embayments. Sediments from these sites exhibited a wide range in concentrations of xenobiotic compounds (e.g. aromatic hydrocarbon (PAH) concentrations ranged from 20 to 50 000 ng/g dry wt. and polychlorinated biphenyl (PCB) concentrations ranged from 2 to 1400 ng/g dry wt.), with the sites in Boston Harbor and Raritan Bay the most heavily contaminated. The following parameters associated with ovarian development were measured: ovarian developmental stage, ovarian atresia, gonadosomatic index, plasma estradiol, fecundity and egg weight. Contaminant exposure was assessed by measuring concentrations of fluorescent aromatic compounds (FACs) in the bile, hepatic aryl hydrocarbon hydroxylase (AHH) activity, concentrations of PCB in liver, ovary and brain and concentrations of xenobiotic-DNA adducts in liver tissue. Additionally, liver tissue was examined histologically for the presence of suspected toxicopathic lesions. In general, indicators of contaminant exposure were elevated and prevalences of suspected toxicopathic lesions were highest in fish from sites within Boston Harbor and Raritan Bay. Hepatic AHH activity, however, was heavily influenced by the reproductive cycle and showed little correlation with other indicators of contaminant exposure. Evidence of decreased egg weight and increased atresia in fish with high tissue concentrations of PCB or high levels of FACs in bile was observed. However, contaminant exposure had no clear negative impact on gonadal recrudescence, gonadosomatic index, plasma estradiol concentrations, or fecundity in female winter flounder. These results are in contrast to results with another Pleuronectid species, English sole (Pleuronectes vetulus), which shows inhibited gonadal development and lower plasma estradiol concentrations at contaminated sites in Puget Sound, WA. The apparent difference between English sole and winter flounder in susceptibility to contaminant-induced reproductive dysfunction could be related to a number of factors, including the differences in the migratory behavior of the two species during the reproductive season. English sole reside in contaminated estuaries throughout vitellogenesis and move offshore to spawn, while winter flounder often remain offshore for extended periods during early vitellogenesis and move into contaminated estuaries prior to spawning. Because of these contrasting migration patterns, both the duration and timing of exposure to contaminants during gonadal recrudescence may differ substantially in these two species and may contribute, in part, to the observed differences in the prevalence of reproductive dysfunction in fish from contaminated estuaries.

Field studies of reproductive success and bioindicators of maternal contaminant exposure in English sole ( Parophrys vetulus)

Our studies have shown that early stages of the reproductive process in female English sole ( Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by measuring levels of polychlorinated biphenyls (PCBs) in ovarian and hepatic tissue, biliary levels of fluorescent aromatic compounds (FACs), and hepatic aryl hydrocarbon hydroxylase (AHH) activities. Additionally, the presence of significant liver disease or ovarian atresia was documented, and levels of plasma estradiol were measured. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining both egg and larval viability. Hepatic AHH activity showed a slight negative correlation with fertilization success, explaining only 4% of the variability. Increased levels of PCBs in ovaries of spawning female English sole were not associated with reduced reproductive success. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

Pesticide-induced atresia in ovary of a fresh water fish, Colisa lalia (Hamilton-Buchanan).

Pesticide-induced atresia in ovary of a fresh water fish, Colisa lalia (Hamilton-Buchanan).

Ovarian Steroid Production in Vitro During Gonadal Regression in the Turkey. II. Changes Induced by Forced Molting1

In the turkey, the onset of incubation behavior is associated with altered ovarian steroidogenesis, ovarian regression, decreased, LH secretion, and increased serum prolactin (Prl) levels. To clarify the relative contribution of circulating LH and Prl to the initiation of ovarian regression, laying hens were exposed for 0, 3, 7, or 14 days to a forced molting procedure (exposure to reduced day length of 6L:18D and removal of feed and water for the initial 3 days) that induces ovarian regression and decreased LH levels but does not increase Prl levels. On each of these days, hens were killed and granulosa and theca interna cells from the largest (F1) and fifth largest (F5) preovulatory follicles and total cells from the small white follicles (SWF) were incubated for 5 h in the presence or absence of ovine LH (oLH; 0-1,000 ng/ml). Force-molted hens exhibited diminished levels of circulating LH, Prl, progesterone (P), androgen (A), and estradiol (E) by Day 3 of treatment. Ovarian atresia began in F1 by the third day of treatment, and included F1 and F5 by the seventh day. No preovulatory follicles were present on the fourteenth day. With both F1 and F5 granulosa cells, production of P in the presence of oLH was initially enhanced (Day 3) and later absent (Day 7). In contrast, production of A by F5 theca interna cells in the presence of oLH was initially suppressed (Day 3) and then returned to pretreatment levels (Day 7).(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian atresia and sex ratio imbalance in white sucker,Catostomus commersoni

In a small north‐temperate lake, 53–56% of post‐spawning, female white suckers spanning a wide range of ages showed ovarian atresia in 1981 and 1987. Comparison of ovarian atresia frequencies and adult sex ratios in nine white sucker populations indicated that this spawning disorder may have been due to a paucity of males at spawning. Adult female:male sex ratio in the King Lake population was 2.82:1 in 1981 and 2.73:1 in 1987, compared with 0.91:1 to 1.75:1 in populations that exhibited lower resorption frequencies (0–7.1%). Removal of 29% of the King Lake male suckers in 1988 elevated the female: male sex ratio to 3.5:1 and frequency of ovarian atresia rose to 75%. Unspawned ova were almost completely resorbed 3 months after the spring spawning period. Post‐spawning mortality was not different between spent and resorbing females. The somatic condition of resorbing and spent females did not differ. There was no evidence that atresia interfered with gamete development subsequently. The sex ratio imbalance present in King Lake was not a function of population density.

Association of the immune system related cells with the human ovarian follicle growth and atresia

Association of the immune system related cells with the human ovarian follicle growth and atresia

Contaminant Effects on Ovarian Development in English Sole (Parophrys vetulus) from Puget Sound, Washington

In a study evaluating the effects of exposure to xenobiotic compounds on ovarian development in English sole (Parophrys vetulus), prespawning females were sampled from four sites in Puget Sound, Washington, during the 1986 and 1987 spawning seasons. Two sampling sites had high concentrations of xenobiotic compounds in the sediment, while the other sites were less contaminated. The following factors associated with ovarian maturation were measured: ovarian developmental stage, ovarian atresia, gonadosomatic index, plasma estradiol, and plasma vitellogenin as estimated from alkali-labile phosphorus. Contaminant exposure was assessed by measuring concentrations of fluorescent aromatic compounds in the bile, hepatic aryl hydrocarbon hydroxylase (AHH) activity, and hepatic polychlorinated biphenyl levels, and liver tissue was examined histologically for the presence of suspected toxicopathic lesions. Female English sole from the heavily contaminated sites were significantly less likely to undergo gonadal recrudescence and had lower mean levels of plasma estradiol than females from the less contaminated sites. The risk of inhibited gonadal recrudescence was significantly increased in sole with elevated hepatic AHH activity, and AHH activity was also significantly negatively correlated with plasma estradiol level. These findings suggest that contaminant exposure may interfere with ovarian development in female English sole.

Luteal morphology, atresia, and plasma progesterone concentrations during the reproductive cycle of two oviparous lizards, Crotaphytus collaris and Eumeces obsoletus.

From ovulation to oviposition, the corpora lutea of the oviparous lizards Crotaphytus collaris and Eumeces obsoletus exhibit three stages of luteal development: 1) luteogenesis, 2) luteal maturity, and 3) luteal regression. Each stage exhibits distinct characteristics, involving changes in: 1) luteal volume, 2) nuclear diameter of cells within the luteal cell mass, and 3) thecal development. Plasma progesterone concentration is greatest during luteogenesis and is positively correlated with ovarian atresia, although atresia occurred throughout the period of gravidity. These data suggest that in these two species, the corpora lutea secrete high amounts of progesterone immediately following ovulation and exhibit morphologically distinct stages of growth and regression.