The Uptake of Tritiated Uridine and Phenylalanine by the Ovaries of Rats and Monkeys

1969 ◽  
Vol 4 (3) ◽  
pp. 655-675
Author(s):  
T. G. BAKER ◽  
HEATHER M. BEAUMONT ◽  
L. L. FRANCHI
Keyword(s):  
Electron Microscope ◽  
Ovarian Tissue ◽  
Ultrastructural Level ◽  
Neonatal Rat ◽  
General Distribution ◽  
Synthetic Activity ◽  
Primordial Follicles ◽  
Ovarian Stroma ◽  
Morphological Identity ◽  
Silver Grains

Rats and monkeys (Macaca mulatta, M. irus) were injected with tritiated uridine or phenylalanine and ovarian tissue was recovered at intervals from 15 min to 24 h later. Autoradiographs were prepared and studied by light microscopy; in addition some specimens which received uridine were examined using the electron microscope. The two isotopes are taken up by the ovaries of both species. The trace obtained with phenylalanine shows a very general distribution, whereas uridine seems to be more selectively incorporated by the various components of the ovary. The nuclei of primordial and growing oocytes are labelled with uridine, and so are the granulosa cells in follicles at all stages of development. The theca surrounding multilayered and vesicular follicles also shows a trace which appears heavier than that over the ovarian stroma. In the neonatal rat, the nuclei of oocytes at the pachytene and diplotene stages take up uridine. Autoradiographs prepared from ovaries fixed from 30 min to 4 h after injection and examined under the electron microscope show silver grains associated with the nucleoli and electron-dense chromosomal cores which are characteristic of these stages in meiotic prophase. Oocytes in primordial follicles, in immature and mature rats, are also labelled, showing that nuclear uptake continues as oocytes enter the ‘resting’ or dictyate stage. Since the chromosomes lose their morphological identity at this time, however, silver grains appear to be randomly distributed over the nuclear matrix. Primordial oocytes in the monkey also have a heavy nuclear label, seen under the light microscope to be associated with chromosomes and nucleoli. At the ultrastructural level, most of the extra-nucleolar silver grains are associated with condensed fibrillar material thought to represent part of the lateral component of chromosomes which are of the ‘lampbrush’ type. The synthetic activity of germinal and somatic elements in the ovary is discussed, and possible differences in the structure and metabolic activity of oocyte chromosomes are considered in relation to intra- and inter-specific variations in radiosensitivity.

Mouse ovarian tissue vitrification on copper electron microscope grids versus slow freezing: a comparative ultrastructural study

2015 ◽  
Vol 27 (7) ◽  
pp. 1020 ◽  
Author(s):  
Ferda Topal-Celikkan ◽  
Sinan Ozkavukcu ◽  
Deniz Balci ◽  
Sibel Serin-Kilicoglu ◽  
Esra Atabenli-Erdemli
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Cancer Therapy ◽  
Ovarian Tissue ◽  
Light And Electron Microscopy ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Primordial Follicles ◽  
User Friendly

There are many reasons, including cancer therapy, for premature ovarian failure and infertility. Oocyte, embryo and ovarian cryopreservation are current options for fertility preservation. Ovarian tissue cryopreservation is essential in patients whose cancer therapy cannot be delayed, including prepubertal girls, and is mostly performed using slow freezing. In the present study, mouse ovarian tissues were vitrified on copper electron microscope grids (n = 18) or conventionally slow frozen (n = 18). Post-thaw tissues were examined histologically using light and electron microscopy and compared with the control group. According to light microscopy observations, antral follicles were found to be better preserved with the slow freezing technique rather than vitrification. Electron microscopy revealed swollen mitochondria in the oocyte cytoplasm, condensations in the zona pellucida, breakages in the junctions of granulosa cells and vacuolisation in the extracellular space in pathologic follicles, which were relatively more frequent, in the vitrification group after thawing. These results indicate that ovarian slow freezing is preferable than vitrification on copper electron microscope grids, especially for larger follicles. Conversely, vitrification of ovarian pieces using cooper grids is user-friendly and provided good protection for primordial follicles and stromal cells. There is a need for further studies into advanced tissue vitrification techniques and carriers.

Ultrastructure of polytene chromosomes of Drosophila isolated by microdissection

1980 ◽  
Vol 45 (1) ◽  
pp. 15-30
Author(s):  
M.R. Mott ◽  
E.J. Burnett ◽  
R.J. Hill
Keyword(s):  
Electron Microscope ◽  
Polytene Chromosomes ◽  
Ultrastructural Level ◽  
Chromosomal Proteins ◽  
Linear Arrays ◽  
Ribonucleoprotein Particles ◽  
Acid Protein

Drosophila polytene chromosomes prepared by a new micromanipulative procedure, which avoids acid squashing, have been examined at the ultrastructural level in the electron microscope. Puffs at 2B, 68C, 74EF, 75B and 85EF, have been examined in some detail, along with the chromocentre and various interbands. The ultrastructure of these chromosomes, which have never been exposed to acid protein denaturants, compares favourably with that of classical acid-fixed specimens. Ribonucleoprotein particles in puffs are seen to be organized in linear arrays and evidence is adduced for looped transcription units. Particles with characteristic sizes and morphologies are observed near the chromocentre, in puffs and in interbands. In interbands RNP particles and ‘superbead’-like chromatin particles may be distinguished. Drosophila polytene chromosomes isolated by micro-manipulation should prove useful for the localization of native chromosomal proteins at an ultrastructural level.

An Ultrastructural and Radioautographic Study of Early Oogenesis in the Toad Xenopus Laevis

1973 ◽  
Vol 12 (1) ◽  
pp. 71-93
Author(s):  
LESLEY WATSON COGGINS
Keyword(s):  
Electron Microscope ◽  
Xenopus Laevis ◽  
Thymidine Incorporation ◽  
Ultrastructural Level ◽  
Extrachromosomal Dna ◽  
Late Pachytene ◽  
Synaptonemal Complexes ◽  
Round Nucleus ◽  
A Cell ◽  
Major Period

Early oogenesis in the toad Xenopus laevis has been investigated at the ultrastructural level, with particular reference to the formation of extrachromosomal DNA. Thymidine incorporation was localized by electron microscope radioautography. In oogonia, the nucleus is irregular in outline and may contain several nucleoli. Oocytes, from premeiotic interphase to late pachytene, are found in cell nests which are estimated to consist of about 16 cells each. Adjacent oocytes within a nest are connected by intercellular bridges and develop synchronously. Each premeiotic interphase-leptotene oocyte has a round nucleus which contains one or two centrally located, spherical nucleoli. Electron-microscope radioautography showed that all nuclei in a cell nest incorporate thymidine synchronously during premeiotic S-phase. In zygotene oocytes, axial cores and synaptonemal complexes are observed in the nucleus and abut against the inner nuclear membrane in the region nearest the centre of the cell nest. The nucleolus is still more-or-less round in outline, but is asymmetrically positioned in the nucleus. It lies near the nuclear envelope on the side of the nucleus furthest away from the attachment of the chromosome ends, that is, nearest the outside of the cell nest. Each nucleolus is surrounded by a fibrillar ‘halo’ of nucleolus-associated chromatin into which a low level of thymidine incorporation occurs during zygotene. This is thought to represent the start of the major period of amplification of the ribosomal DNA. Pachytene is characterized by the presence of synaptonemal complexes in the nucleus. The nucleolus becomes very irregular in outline. The fibrillar area around it, which represents the extrachromosomal DNA, increases in size and thymidine is incorporated over the whole of this region. In late pachytene, many small fibrogranular bodies, the multiple nucleoli, are formed in it. The members of a cell nest become separated from one another at this time and begin to develop asynchronously. In diplotene, synaptonemal complexes are no longer observed in the nucleus. The most prominent structures in the nucleus are now the multiple nucleoli, which increase greatly in number in early diplotene. A large increase in cytoplasmic volume occurs and the oocyte grows in size.

scholarly journals In-vitro fragmentation of ovarian tissue activates primordial follicles through the Hippo pathway

2020 ◽  
Vol 2020 (4) ◽  
Author(s):  
C De Roo ◽  
S Lierman ◽  
K Tilleman ◽  
P De Sutter
Keyword(s):  
Tissue Culture ◽  
Ovarian Tissue ◽  
Hippo Pathway ◽  
Primordial Follicle ◽  
Akt Pathway ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
The Hippo Pathway ◽  
Follicle Activation

Abstract STUDY QUESTION What is the role of the Hippo and PI3K/Akt pathway in follicles during ovarian tissue culture in tissue derived from oncological patients and transgender men? SUMMARY ANSWER Results highlight a Hippo pathway driven primordial follicle activation in vitro, predominantly from Day 0 to Day 4. WHAT IS KNOWN ALREADY In-vitro ovarian tissue culture aims at activating and maturing primordial follicles for fertility restoration in patients with a threatened ovarian reserve. Not all patients are eligible for ovarian cortex transplantation and therefore several groups are attempting to culture ovarian tissue in-vitro. Cortex fragmentation disrupts the Hippo pathway, leading to increased expression of downstream growth factors and follicle growth. The PI3K/Akt pathway is considered the intracellular pathway to where different extracellular factors involved in primordial follicle activation in-vivo converge. In order to optimise current ovarian tissue culture models, information on progression of these pathways during tissue culture is mandatory. STUDY DESIGN, SIZE, DURATION The first step of a multistep cortex culture system was performed using 144 ovarian cortex pieces from a total of six patients. Per patient, 24 cortical strips were cultured for 6 days and six pieces per patient were collected for downstream analysis of follicle development and Hippo and PI3K/Akt pathway targets every second day. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian tissue was obtained from oncological (N = 3; 28.67 ± 4.51 years) and transgender (N = 3; 23.33 ± 1.53 years) patients. Follicles were analysed using haematoxylin-eosin staining and pathways were studied using immunohistochemistry and precise follicle excision by laser capture micro-dissection for RT-qPCR analysis. MIQE guidelines for RT-qPCR were pursued. Reference gene selection (GAPDH, RPL3A, 18s rRNA) was performed using GeNorm Reference Gene Selection Kit. Statistical analysis was conducted with IBM SPSS Statistics 23 (Poisson regression, negative binomial regression, ANOVA and paired t-test). MAIN RESULTS AND THE ROLE OF CHANCE Immunohistochemical analysis confirmed a Hippo pathway driven primordial follicle activation due to mechanical manipulation of the cortical strips. Ovarian tissue preparation and culture induced the inhibitory phosphorylated Yes-associated protein (pYAP) to disappear in granulosa cells of primordial follicles on Day 2. The stimulatory YAP on the contrary appeared in primordial granulosa cells over increasing culture days. Looking at the YAP target connective tissue growth factor (CTGF), a significantly up-regulated CTGF was noted in primordial follicles when comparing Day 2 and Day 4 (ratio Day 2/4 = 0.082; P < 0.05), clearly showing an effect on the Hippo pathway in primordial follicles during tissue culture. Follicle classification showed a significant drop in estimated primordial follicle counts in the oncological cohort (−78%; P = 0.021) on Day 2 and in the transgender cohort on Day 4 (−634%; P = 0.008). Intermediate follicle counts showed a non-significant increasing trend to during culture and this follicle recruitment and growth resulted in a significant rise in estimated primary follicle counts on Day 6 in oncological patients (170%; P = 0.025) and, although limited in absolute numbers, a significant increase in secondary follicles on Day 4 (367%; P = 0.021) in the transgender cohort. Subsequent antral follicle development could not be observed. LIMITATIONS, REASONS FOR CAUTION A limitation is the small sample size, inherent to this study subject, especially as a large amount of tissue was needed per patient to reduce inter-patient variation in different downstream analysis techniques. A particular and specific weakness of this study is the inability to include an age-matched control group. WIDER IMPLICATIONS OF THE FINDINGS These findings support an adapted tissue preparation for Hippo pathway disruption and a shorter first phase of tissue culture. This work may also have implications for transplantation of cryopreserved tissue as larger strips (and thus slower burnout due to less Hippo pathway disruption) could be a benefit. STUDY FUNDING/COMPETING INTEREST(S) This research was financially supported by the Foundation Against Cancer (Stichting tegen Kanker, TBMT001816N), the Flemish Foundation of Scientific Research (FWO Vlaanderen, FWO G0.065.11N10) and the Gender Identity Research and Education Society (GIRES) foundation. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

scholarly journals Histological and ultrastructural changes in turkey liver under intensive rear-ing and influence of xenobiotics

2020 ◽  
Vol 22 (99) ◽  
pp. 63-68
Author(s):  
O. М. Shchebentovska ◽  
A. K. Kostyniuk
Keyword(s):  
Energy Content ◽  
Clinical Manifestations ◽  
Subsequent Development ◽  
Fatty Degeneration ◽  
Ultrastructural Level ◽  
High Energy ◽  
Ultrastructural Changes ◽  
Synthetic Activity ◽  
Standard Diet ◽  
Limited Mobility

Liver problems of various etiologies in turkeys have been reported in many countries for the last 20 years. Poultry dies having no clinical manifestations of the disease, and at pathological autopsy, diffuse haemorrhages and marked dystrophic changes of the organ are noted. To date, there are several factors that can cause such changes, these are unbalanced amino-acid feed, insufficient calcium, biotin, selenium, the very high energy content of feed; zootechnical factors – limited mobility of birds due to cage density violations, high temperature; genetic factors – the influence of estrogens; infectious factors – E. coli, Clostridium, and viruses of Picornaviridae family. The article describes the histopathological and ultrastructural changes in the turkey liver under the influence of various factors. The material for the research was obtained from a farm where turkeys of the “Hybrid Converter” cross are grown, same age, fed with a standard diet that changed according to the technological map of cultivation. On the 50th day of life, a pathological autopsy of the dead poultry was performed, pieces of liver were selected for histological and ultrastructural examination. The visual assessment revealed significantly enlarged liver, the colour from dark red to light brown, flabby consistency. In some cases, diffuse fatty infiltrations of hepatocytes were histologically revealed, in other cases, focal necrosis with the growth of the connective tissue and the formation of massive perivascular couplings were registered. Large vacuolar fatty degeneration of hepatocytes with subsequent development of fibrosis indicates chronic intoxication, probably caused by slow breakdown of fatty acids in cells due to insufficient oxidative phosphorylation, as well as reduced levels of lipotropic factors: choline, methionine and the vitamins. At the ultrastructural level, a large number of lipid inclusions of various sizes, dystrophic changes in mitochondria were observed, which indicates a decrease in the synthetic activity of cells.

scholarly journals In vitro follicle culture in the context of IVF

Reproduction ◽  
2018 ◽  
Vol 156 (1) ◽  
pp. F59-F73 ◽  
Author(s):  
Anamaria C Herta ◽  
Francesca Lolicato ◽  
Johan E J Smitz
Keyword(s):  
Fertility Preservation ◽  
Polycystic Ovary ◽  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Assisted Reproduction Techniques ◽  
Realistic Potential ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Extracellular Matrix Components

The currently available assisted reproduction techniques for fertility preservation (i.e.in vitromaturation (IVM) andin vitrofertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistepin vitrosystems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.

P–449 Evaluation of ovarian reserve in female children and adolescents with non-iatrogenic primary ovarian insufficiency to establish criteria for ovarian tissue cryopreservation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Volodarsky-Perel ◽  
M Zajicek ◽  
D Shai ◽  
H Raanani ◽  
N Gruber ◽  
...  
Keyword(s):  
Fertility Preservation ◽  
Ovarian Reserve ◽  
Predictive Value ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Antral Follicle ◽  
Ovarian Tissue Cryopreservation ◽  
Positive Parameter ◽  
Primordial Follicles

Abstract Study question What is the predictive value of ovarian reserve evaluation in patients with non-iatrogenic primary ovarian insufficiency (NIPOI) for follicle detection in ovarian tissue harvested for cryopreservation? Summary answer Ovarian tissue cryopreservation (OTCP) should be considered if patients present at least one of the following parameters: detectable AMH, FSH≤20mIU/ml, detection of ≥ 1 antral follicle. What is known already In pre-pubertal girls suffering from NIPOI, which majorly has a genetic etiology, fertility preservation using OTCP is commonly practiced. When OTCP was performed in an unselected group of children and adolescents with NIPOI, only 26% of them had follicles in ovarian tissue while 74% did not benefit from the surgery. The role of preoperative evaluation of anti-müllerian hormone (AMH) serum level, follicular stimulating hormone (FSH) serum level, and trans-abdominal ultrasound for the antral follicle count to predict the detection of primordial follicles in the harvested ovarian tissue is unclear. Study design, size, duration We conducted a retrospective analysis of all patients ≤ 18 years old who were referred for fertility preservation counseling due to NIPOI at a single tertiary hospital between 2010 and 2020. If initial evaluation suggested a diminished ovarian reserve and at least one positive parameter indicating a follicular activity (AMH > 0.16ng/ml, FSH ≤ 20mIU/ml, detection of ≥ 1 antral follicle by transabdominal sonography), OTCP was offered. Patients with 46XY gonadal dysgenesis were excluded. Participants/materials, setting, methods OTCP was performed laparoscopically in all cases. A fresh sample of cortical tissue was fixed in buffered formaldehyde for histological analysis. The rest of the ovarian tissue was cut into small cuboidal slices 1–2 mm in thickness and cryopreserved. After the serial sections, the histological slides were evaluated for the presence of follicles by a certified pathologist. Follicles were counted and categorized as primordial, primary, and secondary. Main results and the role of chance During the study period, 39 patients with suspected NIPOI were referred to the fertility preservation center. Thirty-seven patients included in the study were diagnosed with Turner’s syndrome (n = 28), Galactosemia (n = 3), Blepharophimosis-Ptosis-Epicanthus Inversus syndrome (n = 1), and idiopathic NIPOI (n = 6). Of 28 patients with Turner’s syndrome, 6 had 45X monosomy, 15 had mosaicism and 7 had structural anomalies in X-chromosome. One patient with gonadal dysgenesis and one with the presence of Y-chromosome in 20% of somatic cells were excluded from the study. OTCP was conducted in 14 patients with at least one positive parameter suggesting ovarian function. No complications of the surgical procedure or the anesthesia were observed. Primordial follicles were found in all patients with two or three positive parameters (100%) and in three of six cases with one positive parameter (50%). In total, of the 14 patients who underwent OTCP with at least one positive parameter, 11 (79%) had primordial follicles at biopsy (mean 23.9, range 2–47). This study demonstrates a positive predictive value of 79% for the detection of primordial follicles in patients who had at least one positive parameter of ovarian reserve evaluation. If two or three parameters were positive, the positive predictive value increased to 100%. Limitations, reasons for caution This study did not examine the negative predictive value of our protocol as OTCP was not recommended in the absence of positive parameters. The future fertility potential of cryopreserved tissue in the population with NIPOI is unclear and should be discovered in further studies. Wider implications of the findings: We suggest the evaluation of ovarian reserve by antral follicles count, AMH, and FSH serum levels prior to OTCP in patients with NIPOI. By recommendation of OTCP only if ≥ 1 parameter suggesting the ovarian function is positive, unnecessary procedures can be avoided. Trial registration number Not applicable

202 EFFECTS OF ASCORBIC ACID ON IN VITRO CULTURE OF CATTLE PREANTRAL FOLLICLES

2010 ◽  
Vol 22 (1) ◽  
pp. 259
Author(s):  
E. R. Andrade ◽  
R. van den Hurk ◽  
L. A. Lisboa ◽  
M. F. Hertel ◽  
F. A. Melo-Sterza ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Immunohistochemical Analysis ◽  
Development Stage ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Ovarian Cortex

The mechanisms that regulate the gradual exit of ovarian follicles from the nongrowing, primordial pool are poorly understood. The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine the effects of this addition on the growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; 1 fragment was immediately fixed in Bouin’s solution (control). The other fragments were cultured for 2, 4, 6, or 8 days on culture plates in minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, BSA, and antibiotics (MEM+) or in MEM+ plus ascorbic acid (5, 25, 50, 100, or 200 μg mL-1). Ovarian tissue was processed for classical histology, TEM, and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Preantral follicles were classified according to their development stage (primordial, intermediate, primary, and secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare percentages of follicles with PCNA-positive granulosa cells. All analyses were done with Statistical Analysis System (SAS Institute, Cary, NC, USA); P ≤ 0.05 was considered significant. Compared with control fragments, the percentage of primordial follicles was reduced (P ≤ 0.05) and the percentage of growing follicles was increased (P ≤ 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (P ≤0.05), but not when cultures were supplemented with 25, 50, and 100 μg mL-1 of ascorbic acid. Ultrastructural and immunohistochemical analysis of ovarian cortical fragments cultured for 8 days, however, showed the integrity and viability of follicles only when fragments were cultured in the presence of 50 μg mL-1 of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg mL-1 not only stimulates the activation and subsequent growth of cattle primordial follicles that are cultured in vitro for 8 days but also safeguards the viability of these preantral follicles. E. R. Andrade and A. A. Alfieri are recipients of the PRODOC/CAPES fellowship.

scholarly journals Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro

2014 ◽  
Vol 66 (2) ◽  
pp. 411-416 ◽  
Author(s):  
G.Q. Rodrigues ◽  
I.M.T. Lima ◽  
R.N. Chaves ◽  
R. Rossetto ◽  
S.L. Costa ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Ovarian Tissue ◽  
Transforming Growth Factor Β ◽  
Preantral Follicle ◽  
Follicle Growth ◽  
Minimum Essential Medium ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Ultrastructural Studies

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture.

Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 537-549 ◽  
Author(s):  
Regislane P. Ribeiro ◽  
Antonia M.L.R. Portela ◽  
Anderson W.B. Silva ◽  
José J.N. Costa ◽  
José R.S. Passos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Tissue ◽  
Follicle Stimulating Hormone ◽  
Primordial Follicle ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Follicle Activation ◽  
Stimulating Hormone

SummaryThis study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml – Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

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