Histological description of aceh cattle ovary cryopreserved by various cryoprotectants

2018 ◽  
Author(s):  
Roslizawaty Roslizawaty ◽  
Syafruddin Syafruddin ◽  
Tongku Nizwan Siregar ◽  
Azrin . ◽  
Zuhrawati Zuhrawati ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Histological Examination ◽  
Zona Pellucida ◽  
Ovarian Follicle ◽  
Cumulus Oophorus ◽  
Theca Externa ◽  
Theca Interna ◽  
Phosphate Buffered Saline ◽  
Better Than

This research aimed to determine Aceh cattle ovarian follicle morphological integrity after vitrified by various cryoprotectants. Cryoprotectants used in this research were 30% ethylene glycol (EG), 30% dimethyl suphocide (DMSO), and combination of 15% EG + 15% DMSO. Prior to vitrification process, ovaries were cleansed by phosphate buffered saline (PBS) and then cut into ±1 mm³. Ovaries were consecutively submerged into the following liquid for 5 minutes each: PBS+ 0.25 M sucrose; PBS+ 0.5 M sucrose; PBS+ 0.5 M sucrose + 10% cryoprotectants; and PBS+ 0.5 M sucrose + 30% cryoprotectants. Then, ovaries were packed into straws with ±7 cm in length and ± 6 mm in diameter. Before kept in liquid nitrogen, ovaries were first exposed to nitrogen fume for 10 second. After being stored for 1 day, the ovaries were proceed for histological examination. The result showed that Aceh cattle ovarian follicle after vitrification using 30% EG yields the best morphological integrity. Cumulus oophorus, zona pellucida, granulose cell arrangement, theca interna, and theca externa cells were observed clearer in ovary which was vitrified with 30 % EG than those with 30% DMSO and combination of 15% EG + 15% DMSO. As conclusion, 30% EG was able to protect ovary morphological integrity better than 15 % EG + 15% DMSO and 30% DMSO. Furthermore, combination of 15% EG+ 15 % DMSO was relatively better in protecting ovary follicle morphological integrity compared to 30% DMSO.

Memoirs: The Structure of the Ovary and the Formation of the Corpus Luteum in Hoplodactylus Maculatus, Gray

1940 ◽  
Vol s2-82 (326) ◽  
pp. 337-374
Author(s):  
MARY M. M. BOYD
Keyword(s):  
Corpus Luteum ◽  
Zona Pellucida ◽  
Single Layer ◽  
Small Cells ◽  
Follicular Epithelium ◽  
Male And Female ◽  
Physiological Importance ◽  
Egg Membrane ◽  
Theca Externa ◽  
Theca Interna

The structure of the ovary, including stages in the ripening of the oocytes, is described. A prolonged diplotene stage with ‘lamp-brush’ chromosomes is shown to occur in reptiles, as in other classes of vertebrates with large yolky eggs. The striated layer of the egg membrane is shown to be composed of the same cuticular substance as the zona pellucida. A follicular epithelium composed of three types of cells, later reduced to a single layer of small cells, agreeing with Loyez's observations, is described. A discontinuous theca interna, comparable with that of mammalia, is noted outside the membrana propria of the nearly ripe oocyte. A thin, soft, fibrous shell membrane is formed round the uterine egg and polyspermy occurs. The latebra, and the male and female pronuclei in apposition, are described. The corpus luteum is shown to consist of luteal cells invested by fibroblasts from the theca externa. Septa of fibroblasts are also present, but no blood-vessels. The theca is rich in capillaries. The theca interna plays no part in the development of the corpus luteum. A lipoid secretion, which may be of physiological importance, is formed in it. It is compared with that in Monotremes and Marsupials.

Ultrastructural observations of previtellogenic ovarian follicles of dove

Zygote ◽  
2004 ◽  
Vol 12 (4) ◽  
pp. 285-292 ◽  
Author(s):  
Otilia Zarnescu
Keyword(s):  
Electron Microscopy ◽  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Complex Structure ◽  
Cortical Region ◽  
Multivesicular Bodies ◽  
Coated Pits ◽  
Zona Radiata ◽  
Theca Externa ◽  
Theca Interna

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.

44 THE EFFECT OF SUCROSE CONCENTRATION FOR SINGLE-STEP DILUTION ON THE VIABILITY OF CRYOTOP-VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 115
Author(s):  
S. Kondo ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Sucrose Concentration ◽  
Calf Serum ◽  
Single Step ◽  
Bovine Embryos ◽  
Vitrification Solution ◽  
Phosphate Buffered Saline

The aim of this study was to test sucrose concentrations for single-step dilution on the viability of vitrified in vitro-produced bovine embryos. Blastocysts (n = 173, 7 to 8 days after fertilization) were vitrified using the Cryotop (Kitazato, Tokyo, Japan) method placement by incubating the blastocysts in Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 7.5% ethylene glycol, and 7.5% dimethyl sulfoxide for 3 min and then transferring into vitrification solution (Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5 M sucrose). Each embryo was placed on a Cryotop with minimum volume of vitrification solution, and then the Cryotop was plunged into liquid nitrogen. Total time from placement in vitrification solution to plunging into liquid nitrogen was 1 min. The blastocysts were warmed by incubation in the single-step dilution medium for 5 min [0 M sucrose (n = 42), 0.25 M sucrose (n = 44), 0.5 M sucrose (n = 43), and 1.0 M sucrose (n = 44)] at 38.0°C. After dilution, the embryos were washed in TCM-199 supplemented with 20% calf serum and 0.1 mM β-mercaptoethanol and were cultured for 72 h in the same medium at 38.5°C in an atmosphere of 5% CO2. The rates of re-expanded blastocysts and hatched blastocysts were determined at 24 and 72 h after warming, respectively. Data were analysed using the chi-squared test. The percent of re-expanded blastocysts at 24 h after warming in dilution medium supplemented with any level of sucrose was significantly higher (P < 0.05) than in blastocysts warmed without sucrose (Table 1). The hatched blastocyst rate of embryos at 72 h after warming in dilution medium with 0.5 M sucrose was significant higher than that with no sucrose. There were no differences in hatched blastocyst rates between the sucrose concentrations supplemented to the dilution medium. These results suggest that embryos vitrified by the Cryotop method can be diluted in single-step dilution using 0.25, 0.5, or 1.0 M sucrose supplemented to the medium. Table 1.The effect of sucrose concentration for single-step dilution on the viability of Cryotop vitrified in vitro-produced bovine embryos

scholarly journals Structure-Function Relationships in the Wall of the Ovarian Follicle

1981 ◽  
Vol 34 (4) ◽  
pp. 379 ◽  
Author(s):  
JD O'Shea
Keyword(s):  
Cell Function ◽  
Current Knowledge ◽  
Ovarian Follicle ◽  
Follicular Epithelium ◽  
Future Research ◽  
Seminiferous Tubules ◽  
Steroidogenic Cell ◽  
Theca Externa ◽  
Theca Interna ◽  
Follicular Epithelial

This paper reviews current knowledge of the light and electron microscopic structure of the three layers of the mammalian follicular wall-follicular epithelium (membrana granulosa), theca interna and theca externa-and discusses correlations between structure and function. The ultrastructure of follicular epithelial cells in growing follicles emphasizes their protein synthetic and secretory functions; features suggestive of a major steroidogenic function appear only at later stages. Regional differences in follicular epithelial cell function are probably important, although structurally these cells are relatively homogeneous. Structural diversity is more marked in the thecal layers : differentiation in the theca interna is towards fibrocytic and steroidogenic cell types, while that in the theca externa is towards fibrocytic and myoid types. Adherens and gap junctions are present between cells in all layers; however, tight (occludens) junctions have not been convincingly demonstrated between the cells in any of the three layers. Blood and lymph vessels are confined to the thecal layers. However, follicles possess no structural barrier comparable to that associated with the 'blood-testis barrier', and show a correspondingly greater permeability to large molecules than seminiferous tubules. Interactions between the layers of the follicular wall have not yet been intensively investigated, but are likely to play an important role in follicular function. To date, the best-documented interaction between layers is that described in the 'two-cell hypothesis' of oestrogen production. Some potentially useful directions for future research are proposed.

Large equine blastocysts are damaged by vitrification procedures

1995 ◽  
Vol 7 (1) ◽  
pp. 113 ◽  
Author(s):  
S Hochi ◽  
T Fujimoto ◽  
N Oguri
Keyword(s):  
Ethylene Glycol ◽  
Osmotic Pressure ◽  
Zona Pellucida ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Fetal Bovine ◽  
Phosphate Buffered Saline

Viability following vitrification of equine blastocysts with different sizes was investigated in vitro. Twenty-four blastocysts were classified into three groups according to their diameters (< 200 microns, 200-300 microns and > 300 microns; n = 8 each). The solution used for vitrification was defined as EFS and contained 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose in modified-phosphate-buffered saline (m-PBS). During pretreatment with 20% ethylene glycol in m-PBS for 20 min, the larger blastocysts responded to the osmotic pressure caused by 20% ethylene glycol more slowly than the smaller blastocysts. Single blastocysts were loaded into the EFS in 0.25-mL straws, left to stand for 1 min and vitrified in nitrogen vapour. After thawing for 20 s in water (20 degrees C), a fractured zona pellucida or capsule was seen in: 1 of 8 blastocysts < 200 microns in diameter; 1 of 8 blastocysts 200-300 microns in diameter; and 2 of 8 blastocysts > 300 microns in diameter. When the blastocysts were cultured for 48 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO2 in air, 7 of 8 (88%) blastocysts < 200 microns in diameter and 6 of 8 (75%) blastocysts 200-300 microns in diameter developed with re-expansion of the blastocoele. However, the developmental ability of blastocysts > 300 microns in diameter (2 of 8, 25%) was significantly lower than that of blastocysts < 200 microns in diameter (P < 0.05).

82 VIABILITY OF SHEEP-SKIN FIBROBLASTS AFTER SLOW FREEZING

2013 ◽  
Vol 25 (1) ◽  
pp. 188
Author(s):  
Y. Toishibekov ◽  
N. Belyaev ◽  
H. Blackburn ◽  
R. Tleulieva ◽  
B. Katubayeva ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Culture Medium ◽  
Blue Staining ◽  
Wide Range ◽  
Significant Difference ◽  
Fetal Bovine ◽  
Breeding Pool ◽  
Phosphate Buffered Saline ◽  
Skin Samples

Both tissue and cell cryopreservation can be applied for biodiversity conservation. The proper preservation of tissues and cells from a wide range of animals of different species is of paramount importance, because these cell samples could be used to reintroduce lost genes back into the breeding pool by somatic cell cloning. The aim of this work was to investigate the effect of different cooling rates on viability of frozen–thawed sheep fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors (Table 1). Skin samples collected from 10 adult sheep were cut on small pieces (1 × 1 mm), placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and covered with coverslips followed by incubation at 5% CO2, 95% relative humidity, and 37°C. During culture, fibroblasts left skin samples and proliferated. Culture medium was changed every 4 days. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s phosphate buffered saline + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300g for 10 min. For cryoconservation, fibroblast samples were then diluted at a concentration of 2 × 106 cells mL–1 in DMEM + 20% FBS and 10% dimethyl sulfoxide or 10% ethylene glycol and placed into 0.25-mL plastic straws or 2-mL cryovials. Straws were sealed with modeling clay and maintained at +5°C for 120 min before freezing. Cryopreservation of fibroblasts was carried out by 2 procedures: (1) straws were frozen in programmable freezer Kryo Planer 360-3,5 using the following freezing regimen: +5°C to –40°C at –1°C min–1, –40°C to –85°C at –4°C min–1, and then plunged into liquid nitrogen; (2) cryovials were placed in a Styrofoam box and loaded into a freezer at –70°C for 24 h, and then samples were plunged into liquid nitrogen for storage. Samples were thawed for 1 min in a 37°C water bath. Frozen–thawed samples were diluted with DMEM (1 : 5) and centrifuged at 300g for 7 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 106 cells mL–1. Viability of frozen–thawed fibroblast samples was detected using the Trypan Blue staining method. The values obtained (Table 1) are expressed as mean standard error of the mean (SEM). Statistical analysis was done using Student’s test. Results indicated that there was a significant difference in viability between fresh and cryopreserved fibroblasts. However, there were no differences between the cooling procedures. Importantly, our data suggest that the use of 1.5-M ethylene glycol reduced the toxic elements contained in the cryopreservation solution while maintaining a similar ability to produce viable fibroblasts after cryoconservation. Table 1.Effect of 2 cryoprotectant agent (CPA) on the viability of frozen–thawed ovine fibroblasts

Water accumulation as a function of temperature on specimens in an electron microscope cold stage

1986 ◽  
Vol 44 ◽  
pp. 114-115 ◽  
Author(s):  
M.K. Lamvik ◽  
D.A. Kopf ◽  
S.D. Davilla ◽  
J.D. Robertson
Keyword(s):  
Electron Microscope ◽  
Mass Loss ◽  
Liquid Helium ◽  
Liquid Nitrogen ◽  
Liquid Nitrogen Temperature ◽  
Liquid Helium Temperature ◽  
Helium Temperature ◽  
Cold Stage ◽  
Water Accumulation ◽  
Better Than

Last year we reported1 that there is a striking reduction in the rate of mass loss when a specimen is observed at liquid helium temperature. It is important to determine whether liquid helium temperature is significantly better than liquid nitrogen temperature. This requires a good understanding of mass loss effects in cold stages around 100K.

Reproductive characteristics of the african pygmy hedgehog, atelerix albiventris

Reproduction ◽  
2000 ◽  
pp. 143-150 ◽  
Author(s):  
JM Bedford ◽  
OB Mock ◽  
SK Nagdas ◽  
VP Winfrey ◽  
GE Olson
Keyword(s):  
Fallopian Tube ◽  
Zona Pellucida ◽  
Sperm Head ◽  
Reproductive Characteristics ◽  
Cumulus Oophorus ◽  
The Matrix ◽  
Main Components ◽  
Initial Binding ◽  
Structural Matrix

To obtain further perspective on reproduction and particularly gamete function among so-called primitive mammals presently grouped in the Order Insectivora, we have examined the African hedgehog, Atelerix albiventris, in light of unusual features reported in shrews and moles. Atelerix proves to share many but not all of the characteristics seen in these other insectivores. The penis of Atelerix has a 'snail-like' form, but lacks the surface spines common in insectivores and a number of other mammals. Hedgehog spermatozoa display an eccentric insertion of the tail on the sperm head, and they manifest the barbs on the perforatorium that, in shrews, probably effect the initial binding of the sperm head to the zona pellucida. As a possible correlate, the structural matrix of the hedgehog acrosome comprises only two main components, as judged by immunoblotting, rather than the complex of peptides seen in the matrix of some higher mammals. The Fallopian tube of Atelerix is relatively simple; it displays only minor differences in width and in the arborized epithelium between the isthmus and ampulla, and shows no evidence of the unusual sperm crypts that characterize the isthmus or ampulla, depending on the species, in shrews and moles. In common with other insectivores, Atelerix appears to be an induced ovulator, as judged by the ovulation of some 6-8 eggs by about 23 h after injection of hCG. The dense cumulus oophorus appeared to have little matrix, in keeping with the modest dimensions of the tubal ampulla and, while it was not quite as discrete as that of soricids, it did show the same insensitivity to 0.5% (w/v) ovine or bovine hyaluronidase.

scholarly journals Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mark G Larman ◽  
Courtney B Sheehan ◽  
David K Gardner
Keyword(s):  
Ethylene Glycol ◽  
Zona Pellucida ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Initial Increase ◽  
Cell Stage ◽  
Free Treatment ◽  
Zona Hardening ◽  
Free Media ◽  
Oocyte Freezing

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

scholarly journals Immunoelectron microscopic localization of an oviductal antigen in hamster zona pellucida by use of a monoclonal antibody.

1988 ◽  
Vol 36 (11) ◽  
pp. 1441-1447 ◽  
Author(s):  
F W Kan ◽  
S St-Jacques ◽  
G Bleau
Keyword(s):  
Monoclonal Antibody ◽  
Zona Pellucida ◽  
Preimplantation Embryo ◽  
Protein A ◽  
Cumulus Cells ◽  
Gold Particles ◽  
Antigenic Sites ◽  
Cumulus Oophorus ◽  
Entire Thickness ◽  
Immunocytochemical Method

The zona pellucida is an extracellular matrix of glycoproteins which surrounds the mammalian oocyte and preimplantation embryo. We have recently developed monoclonal antibodies against oviductal zona pellucida of the golden hamster. We applied the post-embedding immunocytochemical method using a monoclonal antibody (IgGl,k) to determine the precise location of antigenic sites in the cumulus oophorus complex of the superovulated hamster. By applying the high-resolution protein A-gold technique, we demonstrated that the sites of immunoreactivity were exclusively in the zona pellucida encompassing the oocyte. Other structures within the oocyte and neighboring cumulus cells were not labeled by gold particles. Moreover, gold particles were evenly distributed throughout the entire thickness of the zona pellucida, indicating that this extracellular layer is at least in part made up of an antigen recognized by the monoclonal antibody that is uniformly distributed in the zona matrix.

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