Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Ovarian structure and batch fecundity in Lophiomus setigerus

The ovarian structure and batch fecundity of anglerfish Lophiomus setigerus were examined from specimens collected in the East China Sea during March 1991 to September 1995. The right and left ovarian lobes were connected at their posterior ends. Stalk-like ovigerous lamellae protruded from the ovarian wall. During the spawning season, gelatinous material was secreted from the epithelia of both the ovigerous lamellae and ovarian wall, and these epithelia showed morphological changes accompanying the ovarian maturation cycle. Tertiary yolk, migratory nucleus, and mature stage oocytes occurred in the ovaries between May and November, when females with postovulatory follicles and developing vitellogenic oocytes were collected also. These results suggested an extended spawning season during which females undergo repeated spawnings. When the most advanced oocytes attained the secondary yolk stage, they formed a batch that separated from the adjacent group of smaller oocytes. Batch fecundity ( F ) in 20 females with secondary yolk stage ovaries was related to total length (L_T, mm) as F= 556.2 L_T^1.157 (300≤L_T≤396).     

LncRNA Gm2044 promotes 17β‐estradiol synthesis in mpGCs by acting as miR‐138‐5p sponge

Long noncoding RNAs (lncRNAs) have been demonstrated to play vital roles in mammalian reproduction. Our previous research revealed that lncRNA Gm2044 is highly expressed in mouse spermatocytes and regulates male germ cell function. The gene annotation database BioGPS shows that Gm2044 is not only highly expressed in testicular tissue but also in ovarian tissue, which suggests that Gm2044 may be involved in female reproductive development. In this study, we confirmed that lncRNA Gm2044 promotes 17β‐estradiol synthesis in mouse pre‐antral follicular granulosa cells (mpGCs). Furthermore, bioinformatics methods, western blot, and the luciferase assay proved that Gm2044 functions as a miR‐138‐5p sponge to inhibit the direct target of miR‐138‐5p, Nr5a1, which enhances 17β‐estradiol synthesis through cyp19a1 activation. Taken together, our results provide an insight into the mechanistic roles of lncRNA Gm2044 for 17β‐estradiol synthesis by acting as competing‐endogenous RNAs to modulate the function of mpGCs. Studying the potential lncRNAs, which regulate estradiol release, will be beneficial for the diagnosis and treatment of steroid hormone‐related disease.     

Ovarian regression and apoptosis in the South American teleost Leporinus taeniatus Lütken (Characiformes, Anostomidae) from the São Francisco Basin

Involution and resorption of both postovulatory and atretic follicles were analysed in piau‐jejo Leporinus taeniatus (Characiformes, Anostomidae) in order to evaluate the role of apoptosis during ovarian regression. Histological and ultrastructural analyses showed hallmarks of apoptosis in the granulosa: aggregation of compacted chromatin against the nuclear envelope, cell shrinkage, surface blebbing, loss of cell adhesion and cell fragmentation into apoptotic bodies. Protein synthesis activity preceded the onset of the cell death. The breakdown of the basement membrane led to the detachment of the granulosa cells into the follicular lumen. TUNEL‐positive reactions were detected in in situ DNA fragmentation of granulosa of both postovulatory and atretic follicles. Apoptosis increased in a time‐dependent manner contributing to reduction of the follicular areas. The apoptotic index (per cent of apoptotic cells) of the granulosa increased in postovulatory follicles soon after spawning, then these follicles degenerated and only remnants were observed at 7 days. In contrast, the granulosa cells reabsorbed the yolk during follicular atresia and the apoptotic index increased only in the late stage of regression. The results indicated apoptosis as the major mechanism to rapidly eliminate postovulatory follicles and being an essential process in the ovarian regression after spawning.     

B(a)P暴露干扰细胞周期调控蛋白影响孕早期小鼠卵巢黄体功能

该研究旨在探讨苯并(a)芘[Benzo(a)pyrene,B(a)P]对孕早期小鼠卵巢黄体功能的影响及机制.体内模型:将昆明小鼠每晚按雌雄3∶1的比例合笼,次晨查得阴栓记为孕第1天(d1);将其随机分为对照组和B(a)P处理组,每日早晨称重后以0.1 mL/10 g动物体质量灌胃给予0.2 mg/(kg·d)的B(a)...     

小鼠卵巢组织的超速冻存法研究

目的　本实验通过对小鼠卵巢组织进行冻存研究 ,掌握卵巢的低温生物学特性 ,摸索出一种简便有效的组织器官冻存法 ,为卵巢移植及器官冷冻提供有用的技术方法。方法　通过对小鼠卵巢组织进行慢速程序法与快速液氮蒸汽法冻存 ,比较分析了不同方法所需保护剂种类、浓度、渗透平衡时间。采用对解冻后卵巢组织超微结构观察、组织化学染色、激素测定及自体、异体移植后动情期的恢复作为评价指标。结果与结论　通过上述实验表明用同种冷冻保护剂 ,液氮蒸汽法冻存的卵巢组织超微结构保存良好 ;组织化学染色示其活性与程序法冻存组织相同 ;自体、异体移植后 ,小鼠动情周期的恢复率及血清雌二醇水平各项指标均与慢速程序法冷冻无显著性差异     

Effects of in vitro growth culture duration and prematuration culture on maturational and developmental competences of bovine oocytes derived from early antral follicles

Bovine ovaries offer a large pool of oocytes that could be used for in vitro production of embryos of genetically valuable animals. The effects of in vitro growth (IVG) culture duration (10, 12, and 14 days) on the viability and growth of bovine oocytes derived from early antral follicles (0.5–1 mm diameter) in this study. In addition, the effect of pre-IVM culture with phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) on nuclear maturation of IVG oocytes was also evaluated. In experiment 1, oocyte viability observed after 10 or 12 days of IVG culture was greater (P < 0.05) than that observed after 14 days of culture. Oocyte diameters and proportions of oocytes at metaphase II stage were greater (P < 0.05) when 12 or 14 days of IVG culture where used when compared with 10 days culture. In addition, the proportion of oocytes at metaphase II stage was greater (P < 0.05) when pre-IVM culture was performed for oocytes derived from 12 and 14 days of IVG culture. When 12 and 14 days of IVG culture followed by pre-IVM culture were compared in experiment 2, cumulus cell membrane integrity was greater (P < 0.05) after 12 days. Blastocyst production rate for oocytes obtained after 12 days of IVG culture (24.5%) was greater (P < 0.05) than for oocytes obtained after 14 days (9.9%). In conclusion, 12 days IVG followed by pre-IVM culture was considered the optimal processing system for bovine oocytes derived from early antral follicles when oocyte viability, diameter, maturation, and development competences were considered.     

Diet‐Dependent Genetic and Genomic Imprinting Effects on Obesity in Mice

Although the current obesity epidemic is of environmental origin, there is substantial genetic variation in individual response to an obesogenic environment. In this study, we perform a genome‐wide scan for quantitative trait loci (QTLs) affecting obesity per se, or an obese response to a high‐fat diet in mice from the LG/J by SM/J Advanced Intercross (AI) Line (Wustl:LG, SM‐G16). A total of 1,002 animals from 78 F₁₆ full sibships were weaned at 3 weeks of age and half of each litter placed on high‐ and low‐fat diets. Animals remained on the diet until 20 weeks of age when they were necropsied and the weights of the reproductive, kidney, mesenteric, and inguinal fat depots were recorded. Effects on these phenotypes, along with total fat depot weight and carcass weight at necropsy, were mapped across the genome using 1,402 autosomal single‐nucleotide polymorphism (SNP) markers. Haplotypes were reconstructed and additive, dominance, and imprinting genotype scores were derived every 1 cM along the F₁₆ map. Analysis was performed using a mixed model with additive, dominance, and imprinting genotype scores, their interactions with sex, diet, and with sex‐by‐diet as fixed effects and with family and its interaction with sex, diet, and sex‐by‐diet as random effects. We discovered 95 trait‐specific QTLs mapping to 40 locations. Most QTLs had additive effects with dominance and imprinting effects occurring at two‐thirds of the loci. Nearly every locus interacted with sex and/or diet in important ways demonstrating that gene effects are primarily context dependent, changing depending on sex and/or diet.     

A review of ovarian follicular cysts in cows, with comparisons to the condition in women, rats and rabbits

Some morphological and endocrinological aspects of cystic follicles in cows, women, rats, and rabbits were reviewed. The etiology of follicular cysts remains a mystery, in part due to the difficulty of studying them during formation, particularly in women and cows. Investigations of the morphological and endocrinological nature of cystic follicles during their formation should enhance our understanding of the factors that cause them. Rats and rabbits may provide useful and convenient experimental models for the study of factors which contribute toward cyst formation.     

TGF‐β1 sustains germ cell cyst reservoir via restraining follicle formation in the chicken

The transforming growth factor β (TGF‐β) superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions. TGF‐β1 and its receptors are highly expressed in the ovarian cells of many species. However, the effect of TGF‐β1 on the capacity of the avian germ cell reservoir remains unknown. In this study, 5‐day‐old chicks were injected with TGF‐β1 (2.5, 12.5, and 62.5 μg/kg body weight) for 3 days to assess the effect of TGF‐β1 on early follicle development. Morphological analysis showed that treatment with TGF‐β1 (12.5 μg/kg) increased the number of germ cell cysts and reduced the number of primordial and growing follicles. The diameter and area of oocytes and follicles were decreased after TGF‐β1 treatment. Immunohistochemical staining of the proliferating cell nuclear antigen revealed that the ratios of the positive somatic and granulosa cells were decreased by 16.2% and 2.48%, respectively. Furthermore, more apoptotic cells were observed in the TGF‐β1 group than those of the control by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we cultured the 5d chicken ovaries for 3 days in vitro and found that treatment with TGF‐β1 (10 ng/mL) manifested similar results as the in vivo experiment. However, the negative effect of TGF‐β1 on early ovary development was rescued by treatment with a TGF‐βR1 inhibitor SD208, resulting in increased expression of steroidogenic enzymes and cell cycle‐regulating proteins. In conclusion, TGF‐β1 could maintain the germ cell reservoir by restraining follicle activation involving reduced cell proliferation and steroidogenic enzymes gene expression at the early stage of ovarian development.     

Vegetation Recovery in Slash‐Pile Scars Following Conifer Removal in a Grassland‐Restoration Experiment

A principal challenge to restoring tree‐invaded grasslands is the removal of woody biomass. Burning of slash piles to reduce woody residues from forest restoration practices generates intense, prolonged heating, with adverse effects on soils and vegetation. In this study, we examined vegetation responses to pile burning following tree removal from conifer‐invaded grasslands of the Oregon Cascades. We quantified the longevity and magnitude of fire effects by comparing ground conditions and the cover and richness of plant species in burn‐scar centers (higher‐intensity fire) and edges (lower‐intensity fire) with adjacent unburned vegetation 7 years after treatment. We interpreted patterns of recovery through the responses of species with differing growth forms, habitat affinities, and clonality. Cover of bare ground remained elevated at the centers, but not at the edges of scars; however, much of this effect was due to gopher disturbance. Total plant cover, consisting entirely of native species, was comparable in and adjacent to scars. However, richness remained depressed at the scar centers. Cover of grass, meadow, and non‐clonal species was comparable in and adjacent to scars, but cover of forb, sedge, residual forest, and clonal species was reduced at the centers. Although scar centers had a simpler community structure (fewer but more abundant species) than the adjacent vegetation, they remained free of exotics and recovered quickly, aided by the soil‐disturbing activities of gophers and the regenerative traits of native, disturbance‐adapted species. Pile burning can be a viable and efficient approach to fuel reduction in the absence of exotics.     

Incidence of atresia or of luteinization without rupture of the dominant ovarian follicle in rhesus monkeys treated with estradiol-17β on day 8 of the menstrual cycle

Previous results have established that 17β-estradiol (E₂) administered in capsules for 24 h on Day 6 of the menstrual cycle results in atresia of the dominant follicle (DF). The present experiment was designed to determine if atresia could be induced similarly as late as Day 8, when the DF is presumably larger, to facilitate biochemical analyses. On the morning of Day 8, laparoscopy was used to confirm the presence of the DF, and 4, 6, or 8 Silastic capsules containing E₂ were placed s.c. for 12 or 24 h. Treatment with E₂ for 12 h resulted in atresia of the dominant follicle: 2/4,4/5, and 2/5 with 4, 6, and 8 capsules, respectively. Increases in luteinizing hormone (LH) usually occurred following removal of E₂ after 12 h; similar increases resulted previously from treatment on Day 6, and are interpreted as the result of removal of E₂-induced negative feedback rather than an effect of positive feedback. In contrast to the efficacy of treatment for 12 h, treatment with E₂ for 24 h resulted in a lower incidence of atresia (2/5,1/6, and 1/4 with 4, 6, and 8 capsules), and premature LH surges were seen during treatment in almost all animals, especially with 6 or 8 capsules, initiating luteinization of the DF without ovulation. Regarding intrafollicular changes, E₂ treatment resulted in a 10-fold reduction in follicular fluid concentrations of estrogen, while progesterone (P) levels were augmented; the output of P by granulosa cells in culture was also enhanced by E₂ treatment. We conclude that the DF in monkeys remains susceptible to the atretogenic effects of E₂ as late as Day 8 of the cycle, but the amount of follicular material recoverable is not increased with the later treatment. Furthermore, the similarity between the luteinized unruptured follicles induced in this study and those occurring spontaneously in women suggests that this model may be valuable in elucidating the factors involved in this source of infertility in women. © 1994 Wiley-Liss, Inc.     

Ovarian growth and folliculogenesis in breeding and nonbreeding females of a social rodent,the Zambian common mole-rat,Cryptomys sp.

Zambian common mole-rats are subterranean rodents that live in families with only one female breeding. Her offspring remain in the parental nest and do not reproduce. Behavioral experiments (Burda, '95) demonstrated that their apparent “sterility” is based on incest avoidance and individual recognition of family members. To elucidate whether some kind of morphologically apparent ovarian suppression still takes place in daughters, ovaries of females of known age, weight, and reproductive histories were examined histologically and morphometrically. The body mass of old females (more than 3 years of age) begins to decrease, and the ovaries seem to begin to atrophy at the age of about 3–6 years. Ovaries in neonates exhibited primordial and primary follicles, sometimes clustered in nests. Ovaries of adult nonbreeding females expressed all stages of the follicular development up to tertiary follicles. Many unruptured luteinized follicles were present, but true corpora lutea as a morphological sign of ovulation were missing. Unruptured luteinized follicles also could be found (additionally to true corpora lutea) in ovaries of breeding females. The number of primordial follicles dropped rapidly during the first 2 years of age; the number of primary, secondary, and tertiary follicles was subject to individual variation; and there was no clear correlation with age or reproductive status. While a tendency to form accessory unruptured luteinized follicles may just reflect taxonomic affinities of bathyergids to hystricomorphs, the otherwise complete folliculogenesis in “sterile” daughters and the presence of unruptured luteinized follicles even in breeding females are further evidence that there is no hormonal suppression of the ovarial cycle. We suggest that ovulation in nonbreeding females is not actively suppressed by the breeding female, but instead that it is not released because the triggering mechanisms, most probably repeated copulation, are missing. J. Morphol. 237:33–41, 1998. © 1998 Wiley-Liss, Inc.     

Ovarian maturation and oogenesis in the blue swimmer crab,Portunus pelagicus (Decapoda: Portunidae)

The study was aimed at understanding the process of reproduction and the changes happening in the ovary of Portunus pelagicus during maturation, which would be useful for its broodstock development for hatchery purposes. For that, tissue samples from different regions of the ovary at various stages of maturation were subjected to light and electron microscopy, and based on the changes revealed and the differences in ovarian morphology, the ovary was divided into five stages such as immature (previtellogenic oocytes), early maturing (early vitellogenic oocytes), late maturing (late vitellogenic oocytes), mature (vitellogenic oocytes), and spent (resorbing oocytes). The ovarian wall comprised of an outermost thin pavement epithelium, a middle layer of connective tissue, and an innermost layer of germinal epithelium. The oocytes matured as they moved from the centrally placed germinal zone toward the ovarian wall. The peripheral arrangement of nucleolar materials and the high incidence of cell organelles during the initial stages indicated vitellogenesis I. Movement of follicle cells toward oocytes in the early maturing stage and low incidence of mitochondria and endoplasmic reticulum in the ooplasm during late vitellogenic stage marked the commencement and end of vitellogenesis II, respectively. Yolk granules at various stages of development were seen in the ooplasm from late vitellogenic stage onwards. The spent ovary had an area with resorbing oocytes and empty follicle cells denoting the end of one reproductive cycle and another area with oogonial cells and previtellogenic oocytes indicating the beginning of the next.     

Continuous Covariates in Mark‐Recapture‐Recovery Analysis: A Comparison of Methods

Summary Time varying, individual covariates are problematic in experiments with marked animals because the covariate can typically only be observed when each animal is captured. We examine three methods to incorporate time varying, individual covariates of the survival probabilities into the analysis of data from mark‐recapture‐recovery experiments: deterministic imputation, a Bayesian imputation approach based on modeling the joint distribution of the covariate and the capture history, and a conditional approach considering only the events for which the associated covariate data are completely observed (the trinomial model). After describing the three methods, we compare results from their application to the analysis of the effect of body mass on the survival of Soay sheep (Ovis aries) on the Isle of Hirta, Scotland. Simulations based on these results are then used to make further comparisons. We conclude that both the trinomial model and Bayesian imputation method perform best in different situations. If the capture and recovery probabilities are all high, then the trinomial model produces precise, unbiased estimators that do not depend on any assumptions regarding the distribution of the covariate. In contrast, the Bayesian imputation method performs substantially better when capture and recovery probabilities are low, provided that the specified model of the covariate is a good approximation to the true data‐generating mechanism.