Follicle Membrane Research Articles

Ultrastructure of oogenesis in two tropical oviparous Demospongiae (Porifera): Cinachyrella apion and Tethya maza.

Reproduction is a key step for propagation of any species. Consequently, gametogenesis is crucial, as it links one generation to the other. Oogenesis is influenced by different factors, but it is usually related to the quality and quantity of the food and the capacity of the female to convert these resources into egg production. In Demospongiae (Porifera), oocytes vary in several aspects (e.g., origin, size, and vitellogenic pathways). However, data on oocyte morphology is still fragmentary, and the ultrastructural organization of reproductive cells has been investigated only in a few species, mainly of viviparous sponges. Here, we aimed to comprehend the oogenesis of two tropical oviparous demosponges (Cinachyrella apion and Tethya maza) using light and electron microscopy. In both species, oocytes seemed to originate from archaeocytes. Oocytes of C. apion were surrounded by a collagenous matrix and nurse cells containing many lipid vesicles. The increase of biosynthetic organelles, concomitantly with the presence of yolk vesicle in the ooplasm, indicated that the vitellogenesis was carried out through the mixed pathway. The oocytes of T. maza were surrounded by a follicle cell membrane and nurse cells containing yolk vesicles. The absence of characteristic biosynthetic organelles in the egg of this species indicated that vitellogenesis occured through the heterosynthetic pathway. The oogenesis of C. apion is similar to other species of the genus, while the follicle membrane and nurse cells surrounding the oocytes of T. maza are not observed in any other species of Tethya. These accessory cells were considered to have a trophic role during the oogenesis of the studied species. Moreover, the presence of these accessory cells may have ecological significance, as they accelerate the egg's production through trophic support of the growing oocyte.

Bax/Bcl-2 Expression Ratio Analysis of Rat Ovary Vitrified with Date Juice Concentrate as a Natural Extracellular Cryoprotectant.

The use of extremely low temperatures in vitrification is known to cause cryoinjury so that it can trigger the activation of the intrinsic apoptotic pathway, which can damage the structural integrity of the pre-antral follicle. Based on that, it is necessary to use an appropriate cryoprotectant to protect the preserved cell. This study aimed to identify the potential use of date juice concentrate (DJC) as a natural extracellular cryoprotectant to suppress the rate of apoptosis after vitrification. This experimental research uses 24 samples of ovarian rats. Rats were fed and drank an ad libitum. Ovaries were isolated in the proestrus phase, then processed into slides for immunohistochemistry (IHC) staining using anti-Bax and anti-Bcl-2 antibodies. IHC results were evaluated for the brown colour using ImageJ IHC Profiler. The results were analysed as an optical density and displayed in the Bax/Bcl-2 ratio. All data were statistically analysed with either parametric (analysis of various) or non-parametric (Kruskal-Wallis) tests. The combination of EG 7.5% + DJC 15% (KP2) showed the lowest Bax/Bcl-2 ratio in primordial and primary follicles. Meanwhile, the lowest Bax/Bcl-2 ratio in secondary follicles is found in KP4 (EG 15% + DJC 15%). The DJC is known to contain a dominant amount of glucose. The DJC shows antioxidant activity and contains antioxidant compounds, phenols and flavonoids. The sugar content and antioxidant compounds of DJC can protect against follicle membrane damage, so the rate of intrinsic apoptosis pathway is also suppressed initially with Bax protein suppression in the mitochondrial membrane.

Research Note: Integrated transcriptomic and metabolomic analysis reveals potential candidate genes and regulatory pathways associated with egg weight in ducks

Egg weight is an important indicator of egg phenotypic traits, which directly affects the economic benefits of the poultry industry. In the present research, laying ducks were classified into high egg weight (HEW) and light egg weight (LEW) groups. To reveal the underlying mechanism that may be responsible for the egg weight difference, the integrated analysis of transcriptomes and serum metabolomics was performed between the two groups. The results showed extremely significant differences (P < 0.01) in the total egg weight at 300 d, and average egg weight between the HEW and LEW groups. 733, 591, 82, and 74 differentially expressed genes (DEGs) were identified in the liver, magnum, F1, and F5 (hierarchical follicles) follicle membrane, respectively. The candidate genes were screened further from the perspective of forming an egg. In terms of egg yolk formation, the functional analysis revealed fatty acid metabolism-related pathways account for 36% of the liver's top pathways, including fatty acid biosynthesis, folate biosynthesis, fatty acid metabolism, and glycerol lipid metabolism pathways. FASN gene was identified as the key candidate gene by comprehensive analysis of gene expression and protein-protein interaction (PPI) network. In the follicle membrane, the DEGs were mainly enriched in protein processing in the endoplasmic reticulum, and MAPK signaling pathway, and HSPA2, HSPA8, BAG3 genes were identified as crucial candidate genes. In terms of egg white formation, the functional analysis revealed protein metabolism-related pathways account for 40% of the magnum's top pathways, which includes protein processing in the endoplasmic reticulum pathway. HSP90AA1 and HSPA8 genes were identified as key candidate genes. In addition, the integrated transcriptomic and metabolomic analysis showed that arginine and proline metabolism pathways could contribute to differences in egg weight. Thus, we speculated that the potential candidate genes, regulatory pathways, and metabolic biomarkers mentioned above might be responsible for the egg weight difference. These findings might provide a theoretical basis for improving the egg weight of ducks.

Transcriptomic analyses of the HPG axis-related tissues reveals potential candidate genes and regulatory pathways associated with egg production in ducks

BackgroundEgg production is one of the most important economic traits in the poultry industry. The hypothalamic-pituitary–gonadal (HPG) axis plays an essential role in regulating reproductive activities. However, the key genes and regulatory pathways within the HPG axis dominating egg production performance remain largely unknown in ducks.ResultsIn this study, we compared the transcriptomic profiles of the HPG-related tissues between ducks with high egg production (HEP) and low egg production (LEP) to reveal candidate genes and regulatory pathways dominating egg production. We identified 543, 759, 670, and 181 differentially expressed genes (DEGs) in the hypothalamus, pituitary, ovary stroma, and F5 follicle membrane, respectively. Gene Ontology (GO) analysis revealed that DEGs from four HPG axis-related tissues were enriched in the "cellular component" category. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the neuroactive ligand-receptor interaction pathway was significantly enriched based on DEGs commonly identified in all four HPG axis-related tissues. Gene expression profiles and Protein–Protein Interaction (PPI) network were performed to show the regulatory relationships of the DEGs identified. Five DEGs encoding secreted proteins in the hypothalamus and pituitary have interaction with DEGs encoding targeted proteins in the ovary stroma and F5 follicle membrane, implying that they were these DEGs might play similar roles in the regulation of egg production.ConclusionsOur results revealed that neuroactive ligand-receptor interaction pathway and five key genes(VEGFC, SPARC, BMP2, THBS1, and ADAMTS15) were identified as the key signaling pathways and candidate genes within the HPG axis responsible for different egg production performance between HEP and LEP. This is the first study comparing the transcriptomic profiles of all HPG axis-related tissues in HEP and LEP using RNA-seq in ducks to the best of our knowledge. These data are helpful to enrich our understanding of the classical HPG axis regulating the egg production performance and identify candidate genes that can be used for genetic selection in ducks.

123 Development of cryopreservation protocol for zebrafish ovarian fragments using controlled slow cooling

Cryopreservation of gametes is an important method of conservation of germplasm and has wide range of applications in aquaculture and fisheries management. Cryopreservation is the most efficient method for long term storage of genetic materials. Though fish sperm cryopreservation has been a success, developing successful protocols for eggs and embryo cryopreservation still remain elusive. Several studies have been undertaken on cryopreservation of isolated zebrafish ovarian follicles at different stages yet the protocols used lead to compromised viability. Cryopreservation of follicles within ovarian tissue has attracted considerable attention in recent years. It has become a valid alternative to cryopreservation of human oocytes. The present study was undertaken to determine the effect of cryopreservation on zebrafish ovarian tissue fragments containing the stage I and stage II follicles. The effect of cryopreservation medium, the effect of cryoprotectants, cooling rate and ovarian follicle viability assessment after controlled slow cooling were investigated. Ovarian follicle membrane integrity was assessed by trypan blue (TB) and fluorescein acetate-propidium iodide (FDA-PI) staining. ATP levels were measured to assess the metabolic status following cryopreservation. In these experiments ovarian tissue fragments were exposed to cryoprotectant solutions for 30 min at 22 °C and then were loaded into 0.5 ml plastic straws before placing in a programmable cooler (Planer KRYO 550). Ovarian tissue fragments incubated in cryoprotectant-free L-15 medium or KCl buffer were used as controls. The following cooling protocol was used: cooling at 0.3 °C/min, 0.4 °C/min, 1 °C/min, 2 °C/min, 4 °C/min and 7 °C/min from 20 °C to seeding temperature (−7.5 °C for 2 M methanol). Manual seeding and hold for 5 min, freezing from seeding temperature to −40 °C at 2 °C/min, from −40 °C to −80 °C at 10 °C/min and hold for 10 min, samples were then plunged in liquid nitrogen at −196 °C and held for at least 10 min. Samples were thawed using a water bath at 28 °C. Removal of cryoprotectant was conducted in four steps. The results from the present study indicated that the tissue fragments can be incubated in 2 M methanol + 20% FBS in L-15 medium for 30 min at room temperature and then frozen to −196 °C at post-seeding cooling rate 4 °C/min and the removal of the cryoprotectant in four steps. After cryopreservation the survival rate of the follicles was assessed using TB staining, FDA + PI staining and ATP assay. The survival rate after the cryopreservation procedure were 55.4 ± 2.3% for stage I and 68.2 ± 1.9% for stage II using TB staining and 48.2 ± 2.9% for stage I and 54.2 ± 2.6% for stage II follicles using FDA + PI staining. The results obtained from ATP assay showed compromised survival of the ovarian follicles. The present study also indicated that the use of non-permeating cryoprotectants is not beneficial for the zebrafish ovarian tissue cryopreservation since it decreased the survival rate of the follicles when compared to the use of permeating cryoprotectants. The results obtained after the in-vitro culture of the cryopreserved fragments showed that there was no growth of follicles obtained after in-vitro culture treatment and the survival rate of the cryopreserved follicles were significantly lower to those of the non-cryopreserved follicles. The results obtained in this study provide useful information for future cryopreservation protocol development. Source of funding: Funding support for the programme of research was provided by the strategic research funds of the iBEST, University of Bedfordshire Conflict of interest: None declared. tzhang@bournemouth.ac.uk

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles

Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.

Histological study on the gonadal development of Scatophagus argus

The fish spotted scat Scatophagus argus,as a new resource of marine aquaculture,is popularized in South China in recent years.Studies on the gonadal development can provide the theoretical knowledge for the artificial propagation of Scatophagus argus.Generally,after two years of growth,some individuals can reach sexual maturation in the south of China.In this study,histological observation of gonads was performed based on paraffin section technique and HE staining method.The linear alignments of tubular structures are observed in the testis,which indicates that the testis of the Scatophagus argus belongs to the tubular type.The development of testis may be divided into 5 stages,i.e.multiplication of spermatogonial cells,growth of spermatocytes,maturation of spermatocytes,emergence of spermatoblasts,maturation of spermatozoa,etc.The average gonado somatic index of the testis was between 0.2% and 1.5%.The gonado somatic index(GSI)reached the peak at the stageⅤ,and the the peak of hepato somatic index(HSI)was observed at the stage Ⅳ.The oocyte deveolpment was divided into 5 phases and ovary development consisted of 5 stages accordingly.The average gonado somatic index of the testis was between 1.2% and 14.5%.The gonado somatic index(GSI)reached the peak value at the stage Ⅴ and the peak of hepato somatic index(HSI)was found in stage Ⅳ.The yolk nucleus and follicle membrane are observed at phase Ⅱ of oocyte.The number of yolk granules and oil droplets increased rapidly at the phaseⅢ,and subsequently yolk granules fused into pieces at the phase Ⅳ Apart from a large number of oocytes at the phase Ⅴ,there are still many oocytes of phase Ⅱ,Ⅲ and Ⅳ in ovaries at the stage Ⅴ.The similar condition was also found in ovaries after spawning.It indicates that oocyte development in Scatophagus argus is asynchronous.

The gonadotropin receptors FSH-R and LH-R of Atlantic halibut (Hippoglossus hippoglossus)—2. Differential follicle expression and asynchronous oogenesis

The biological activity and spatio–temporal expression patterns of the gonadotropin receptors FSH-R and LH-R were examined in the repetitive spawner Atlantic halibut to elucidate the gonadotropic regulation of the asynchronous follicle development. The cloned receptors were expressed in mammalian COS-7 cells, and stimulation with sea bass FSH and LH increased the cAMP production. The halibut FSH-R and LH-R genes were shown to be highly expressed in the gonads of sexually mature fish, but the transcripts were also found in extra-gonadal tissues such as pituitary and brain. Different expression patterns of FSH-R and LH-R in the developing follicles were documented by semi-quantitative RT-PCR. Abundant FSH-R mRNA was found in the small follicles during primary growth and vitellogenesis, and the signals were localized to the granulosa cells by in situ hybridization. In contrast, follicular LH-R mRNA was hardly detectable during the early stages. Conversely, in follicles during final maturation FSH-R mRNA levels tended to decrease, while the expression of LH-R was highly upregulated. Whereas the pituitary FSH and LH are asynchronously expressed in annual spawners, both gonadotropins were expressed in the female halibut pituitary throughout the reproductive cycle, except in the prespawning females. Hence, the sequential gonadotropic activation of ovarian follicle growth and maturation in repetitive spawners is probably regulated by modulating the temporal expression of FSH-R and LH-R in the follicle membrane.

Avian apolipoprotein A-V binds to LDL receptor gene family members

Apolipoprotein A-V (apoA-V) affects plasma triglyceride (TG) levels; however, the properties of apoA-V that mediate its action(s) are still incompletely understood. It is unclear how apoA-V, whose plasma concentration is extremely low, can affect the pronounced TG differences observed in individuals with various apoA-V dysfunctions. To gain novel insights into apoA-V biology, we expanded our previous studies in the chicken to this apolipoprotein. First, we characterized the first avian apoA-V, revealing its expression not only in liver and small intestine but also in brain, kidney, and ovarian follicles and showing its presence in the circulation. Second, we demonstrate directly that galline apoA-V binds to the major LDL receptor family member (LR) of the laying hen and that this interaction does not depend on the association of the apolipoprotein with lipid or lipoproteins. We propose that a direct interaction with LRs may represent a novel, additional mechanism for the modulation of TG levels by apoA-V.

A radioligand binding assay to measure anti-thyroperoxidase autoantibodies in mice

Autoimmune (Hashimoto's) thyroiditis is a chronic inflammatory disease which affects > 3% of the population and shows an increasing prevalence with increasing age. Anti-thyroid autoantibodies, particularly against thyroperoxidase (also known as thyroid peroxidase or TPO), occur commonly in humans with autoimmune thyroid disease, and assays for anti-TPO autoantibodies are used in clinical diagnosis. In contrast anti-TPO autoantibodies have not been observed in classical mouse models of autoimmune thyroiditis, except in cases where mice were deliberately immunized with TPO. In the past, detection of anti-TPO autoantibodies in mice has relied on an indirect immunofluorescence assay (iIFA) which screens for thyroid follicle membrane staining in frozen sections of mouse thyroid glands. Since recent transgenic mouse models of autoimmune thyroiditis spontaneously develop anti-TPO autoantibodies, an assay other than serial dilution and iIFA would be useful to detect and quantify these autoantibodies. In this paper we describe such an assay, based on the capacity of autoimmune mouse sera to bind to the extracellular domain of mouse TPO which was produced in a radioactively labeled form using a coupled in vitro transcription/translation system. The same approach, using human TPO, could provide a highly sensitive method to detect anti-TPO autoantibodies in humans.

Permeability Characteristics of Ovine Primordial Follicles Calculated with Two Parameter Kedem-Katchalsky Formulation

The osmotic responses of isolated ovine primordial follicles when suspended in various concentrations of several cryoprotective additives (CPAs) have recently been published. We have used the Kedem-Katchalsky formulation, written with two parameters as variables, to calculate permeability of the follicle membrane to water, Lp, and permeability to CPAs, ω. By fitting the model to the osmotic responses of follicles determined by Amorim and colleagues, we were able to calculate the permeability coefficients in the presence of 0.5, 1.0, 1.5, 2.0, or 2.5 M solutions of each of the following CPAs: dimethylsulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH), and glycerol (GLY). In the presence of DMSO, the best-fit values of L p ranged from 0.15 to 0.29 × 10-14 m 3/N-sec, and ω ranged from 0.30 to 0.50 × 10 -11 mol/N-sec. In the presence of EG, the corresponding values of Lp ranged from 0.25 to 0.47 × 10-14 m 3/N-sec, and ω ranged from 0.20 to 0.65 × 10 -11 mol/N-sec. In the presence of PROH, Lp ranged from 0.10 to 0.62 × 10-14 m3/N-sec, and ω ranged from 0.10 to 0.40 × 10-11 mol/N-sec. And finally, in the presence of GLY, the best-fit values of Lp ranged from 0.19 to 0.32 × 10-14 m3/N-sec, and ω ranged from 0.05 to 0.32 × 10-11 mol/N-sec. The cross correlation or the reflection coefficient, σ, defined as σ = 1 - ωυCPA/ Lp, where υCPA is the partial mole volume of the CPA, ranged from 0.763 to 0.926 in the presence of DMSO, from 0.870 to 0.977 in the presence of EG, from 0.813 to 0.983 in the presence of PROH, and from 0.918 to 0.988 in the presence of GLY. These values of Lp and ω (and σ) can be used to predict the volumetric response of ovine follicles under arbitrary CPA loading and unloading conditions and consequently, the presumptive optimal conditions. © Mary Ann Liebert, Inc.

Design and fabrication of a micro thermal actuator for cellular grasping

The development of a novel polymer-based micro robotic gripper that can be actuated in a fluidic medium is presented in this paper. Our current work is to explore new materials and designs for thermal actuators to achieve micromanipulation of live biological cells. We used parylene C to encapsulate a metal heater, resulting in effectively a tri-layered thermal actuator. Parylene C is a bio-compatible dielectric polymer that can serve as a barrier to various gases and chemicals. Therefore, it is suitable to serve as a thermal/electrical/chemical isolation material for protecting the metal heater from exposing to an aqueous environment. We have demonstrated parylene actuators (2 mm×100 μm×0.5 μm) to operate in an aqueous environment using 10 to 80 mW input power. The temperature of these actuators at full deflection was estimated to be ∼60°C, which is much lower than the typical requirement of >100°C to actuate other conventional MEMS actuators.Danio rerio follicles in fluidic medium were captured successfully using these actuators. Moreover, these actuators were found to be responsive to moderate rise in environmental temperature, and hence, we could vary the fluidic medium temperature to actuate trimorphs on a chip without any input of electrical energy, i.e., raising the fluidic temperature from 23°C to 60°C could actuate the trimorphs to grasp follicles of ∼1 mm size in diameter. At 60°C, the embryos inside the follicles were observed to be alive, i.e., they were still moving in the biological fluid isolated by the follicle membrane. The smallest follicles grasped were ∼500 μm in diameter using 800 μm×130 μm×0.6 μm actuators. The fabrication process, modeling, and optimization of the trimorph actuators are presented. Based on the successful operation of these polymer-based actuators, we are currently developing multifinger thermal microgrippers for cellular grasping and manipulation, which can potentially be hybridly integrated with circuits for computer control.

Mechanism of Transovarian Transmission of Salmonella enteritidis in Laying Hens

To understand the mechanism of transovarian transmission of Salmonella enteritidis in laying hens, experiments were conducted to examine the isolation of S. enteritidis from the preovulatory follicles of experimentally infected hens. Salmonella enteritidis was isolated from the preovulatory follicles in 16 birds (from follicle membrane alone in 10 birds, from the follicle yolk alone in 4 birds, and from both membrane and yolk in 2 birds). In addition, 83 S. enteritidis isolates of the major phage types prevalent in United States were tested for attachment to hen ovarian granulosa cells and HEp-2 cells. Salmonella enteritidis demonstrated three different patterns of attachment to granulosa cells, namely, local, diffuse, and aggregative; whereas, only local attachment pattern was observed on HEp-2 cells. The total number of S. enteritidis isolates that demonstrated any pattern of attachment was significantly greater on the granulosa cells than on HEp-2 cells (P < .05). Salmonella enteritidis isolates of phage Types 8 and 28 demonstrated similar patterns of attachment on granulosa cells derived from the mature and developing follicles of the hen ovary. This suggests that S. enteritidis can colonize the preovulatory follicles at different stages of development. Preincubation of bacteria with the tetrapeptide arg-gly-asp-ser, the amino acid sequence known to mediate the interaction of adhesive proteins with cells, abrogated the local attachment of bacteria to granulosa cells. These results suggest that S. enteritidis can colonize the preovulatory follicles by interacting with the ovarian granulosa cells and that adhesive proteins may be involved in this process.

Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout, Oncorhynchus mykiss

The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (125iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (Kd=8.2·10-9 mol·1-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.

Bovine inhibin immediately inhibits the electrophysiological response to chorionic gonadotrophin in ovarian follicles ofXenopus laevis.

Inhibin was originally isolated from ovarian tissue using, as a bioassay, its ability to diminish synthesis and secretion of gonadotrophins in pituitary cells after several days. Inhibin is known also to modify responses to gonadotrophins in the ovary after stimulation times of hours or days. Here we show that action for less than 4 min of purified bovine inhibin on ovarian follicles from Xenopus laevis causes a specific, dose-responsive (greater than 10 U/ml) inhibition of the membrane hyperpolarization produced by stimulation with chorionic gonadotrophin. The CG-induced change in follicle membrane potential has previously been shown to be caused by potassium efflux produced by rises in intracellular cAMP. Inhibin did not affect similar hyperpolarization stimulated by 10 microM adenosine. The apparent Ca(2+)-dependent Cl- currents which are often associated with CG action, were not inhibited by inhibin. At concentrations up to 2000 U/ml, inhibin did not significantly alter the timing of CG-induced germinal vesicle breakdown in the oocytes, which may suggest functional action on other developmental processes.

Transport of 17α,20β-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis ovarian follicles

Extremely low levels of the maturation inducing steroid (MIS) 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP) were found in the ooplasm and ovarian follicle membranes of Atlantic salmonSalmo salar ouananiche, a finding that is at variance with the elevated blood levels of the steroid. The uptake of MIS at physiological concentrations into brook trout follicles occurred by passive diffusion. Uptake of the steroid into the ovarian follicle membrane, consisting of zona radiata and the attached follicle cells, deviated from linearity in a double reciprocal plot. These results suggest that 17α,20β-DHP is binding to a receptor-like protein in the ovarian follicle or the zona radiata membrane surrounding the oocyte, and extend our previous demonstration of 17α,20β-DHP receptor-like activity in the zona radiata membrane of the late stage brook trout oocytes. An oocyte cytoplasmic receptor gave subunits on SDS PAGE that were similar to the membrane and cytosol receptors previously described.

Studies on ligand recognition by vitellogenin receptors in follicle membrane preparations of the german cockroach, Blattella germanica

An in vitro binding assay was used to analyze the interaction between vitellin and its receptor in follicle membrane preparations of the cockroach, Blattella germanica. Binding was found to be optimal at pH 6.8. Specific binding was saturable, and Scatchard analysis yielded a K d-value of 79 nM and a total receptor concentration of 51 fmol/follicle. Glycopeptides and oligosaccharides prepared from vitellin competed for vitellin binding, whereas yeast mannan did not. Glycopeptides digested with α-mannosidase stimulated vitellin binding. Vitellins of the cockroach species Simploce capitata and Nauphoeta cinerea competed for binding of B. germanica vitellin, whereas vitellin of another cockroach, Leucophaea maderae, stimulated binding. Analysis of the oligosaccharide structure of the vitellin in these species revealed differences in the proportions of individual oligosaccharides in the total carbohydrate, however, these differences do not correlate with the ability of the vitellins to compete for binding. The results suggest that the high mannose oligosaccharides of vitellin are necessary but not sufficient for binding to the vitellogenin receptor.

Changes in the pituitary and plasma LH, plasma and follicular progesterone and estradiol, and plasma testosterone and estrone concentrations during the ovulatory cycle of the quail ( Coturnix coturnix japonica)

Luteinizing hormone (LH) and sex steroid hormones in the pituitary, plasma, and hierarchical follicles of the quail maintained under 14L:10D condition were measured during the ovulatory cycle at 2-hr intervals using radioimmunoassay. Pituitary levels of LH during the ovulatory cycle showed two peaks which occurred 16 and 10 hr before ovulation. A significant decrease in LH concentration was observed from 10 to 6 hr before ovulation, shortly after the onset of light (dawn). Plasma LH started to increase from 10 hr before ovulation, and reached a peak 6 hr before ovulation. Plasma progesterone also started to increase shortly after dawn and reached a peak 4 hr before ovulation. A single peak of plasma testosterone was observed 6 hr before ovulation. Two peaks of plasma estradiol concentration were observed at 22 and 6 hr before ovulation. A similar tendency of the change in plasma concentration of estrone to that of estradiol was observed during the ovulatory cycle. In the largest follicle, the progesterone content started to increase from 10 hr before ovulation, reached a peak 2 hr before ovulation and decreased abruptly after the ovulation. The contents of the second to fourth largest follicles were low. These findings strongly suggest that progesterone in the largest follicle is a main cause of ovulation by producing enzymes which act to break the follicle membrane. The estradiol contents of the third and fourth largest follicles were high, and showed the peak at 18 hr before ovulation in the third largest one and at 8 hr before ovulation in the fourth largest one. The contents of the largest, ruptured, and the second largest follicles were low compared with the third or fourth largest one. These results suggest that the onset of light may be a signal for the increases of plasma LH and progesterone and follicular progesterone which are responsible for the induction of ovulation in the quail.

A theoretical consideration of the periodontal membrane as a collagenous thxotropic system and ist relationship to tooth eruption

It is proposed that the physiological behaviour of the dental follicle and perodontal membrane is explicable, on theoretical grounds, as having the characteristics fo a thixotropic gel. If the collagen “fibres” of soft connective tissue as seen in fixed specimens area accepted as the static appearance of a dynamic system, i.e. artefacts of fixation, then the physiologicla behaviour of theis collagenous tissue and the eruption of teeth may be explained in simple terms by changes in the viscosity of this biological system.

家鶏卵巣の血管分布に関する研究

産卵鶏の卵巣には大小多数の卵胞が見られ,とくに成熟卵胞では排卵の数日前より急速に卵黄を蓄積しはじめ,卵胞の直径を増大させる.このように短期日に多量の卵黄を蓄積できるのは,卵胞に関与する血管によるものと考えられる.NALBANDOV and JAMES5)は鶏の成熟卵膜には5層の血管層が分布していると報告している.著者は前報で,卵胞が血管系を獲得する過程について報告し,そのなかの一部に成熟卵胞に分布する血管について述べた6).またBRADLEY and GRAHAME1),千島2),久富3),KAUPP4),STURKIE8),らは卵胞膜に分布する血管について記載している.従来の報告では卵胞膜に分布する血管層の種類と分布位置をほぼ明らかにした程度である.よって本実験は成熟卵胞膜の全域に分布する血管を解明するために,色素注入を行ない,伸展標本,横断切片を作製して各層における分布状態および各層とのつながり,そして動静脈の分布領域について観察した.さらに血管の分布•分岐状態を模型図に表わした.