scholarly journals Follicular Histomorphometry and Evaluation of Ovarian Apoptosis in Queens of Different Age Groups

2018 ◽  
Vol 44 (1) ◽  
pp. 8
Author(s):  
Isadora Resende de Carvalho ◽  
Daniela Scantamburlo Denadai ◽  
Juliane Teramachi Trevisan ◽  
Heloisa Maria Falcão Mendes ◽  
Sílvia Helena Venturoli Perri ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Plasma Concentrations ◽  
Age Groups ◽  
Oocyte Diameter ◽  
Primordial Follicles ◽  
Tunel Reaction ◽  
Area And Perimeter ◽  
Ovulatory Follicles ◽  
The Relationship

Background: In humans and bitchs the age is another factor that may affect the size of ovarian structures, verifying alterations in the quality of the pool and size of follicular structures, which can compromise the use of these structures for in vitro maturation. There are no reports correlating the morphometric characteristics of the follicles and ovarian apoptosis at different ages in cats. The aim of this study was to evaluate the histomorphometric parameters of follicular growth and the relationship with the occurrence of apoptosis in ovarian tissue of young, adult and senile queens.Materials, Methods & Results: Eighteen domestic queens, multiparous, of different breeds and age groups were used in this study and divided into three groups according to their ages: five months to one year - young; (7.8 ± 1.0 months); one to six years - adults (2.8 ± 0.5 years); and more than six years - senile (8.0 ± 0.9 years). Vaginal cytology was performed in order to characterize the estrous phase associated with plasma concentrations of progesterone. The morphology and percentage of the vaginal epithelium cells were evaluated and queens were classified into estrous and non-estrus and plasma concentrations of progesterone were determined. Ovarian samples were collected after ovariohysterectomy to routine histological processin and all follicles were counted and categorized into two groups, non-atresic and atresic. The mean follicular and oocyte diameters were calculated between the measurement of the largest diameter and perpendicular diameter. The relationship between follicle and oocyte were determined using the measurements of diameter, area and perimeter. The apoptotic cells were detected and cells were considered positive when TUNEL reaction was detected. The morphometric index of 1039 follicles were evaluated. Primordial follicles in young animals showed larger diameter, follicular area and perimeter than the structures of adult queens, as well as the unilaminar primary follicles of the same group were larger compared with senile animals (P < 0.05). Comparing adult and younger queens, the first showed a significant decrease of oocyte diameter in primary and unilaminar primary follicles, as well as oocyte area and perimeter (P < 0.05). The values for follicular diameter, oocyte area and perimeter for multilaminar primary, secondary and pre-ovulatory structures did not present statistical differences between the groups (P > 0.05). For the pre-ovulatory follicles there was no positive correlation between the oocyte growths regarding the follicles (P > 0.05). Only in senile animals positive markers for apoptosis were identified in nuclei of primordial follicles. No significant differences concerning the number of follicles and Tunel positive cells were observed between groups (P > 0.05).Discussion: Considering the importance of this study for greater knowledge in the basic aspects for reproductive biotechnologies, we verified that secondary follicles showed the largest diameters and younger animals the largest values for diameter, area and perimeter, suggesting that this age group could be ideal for the use and manipulation of oocytes. The process of follicular atresia is characterized by the occurrence of apoptosis, or programmed cell death when the organism begins to efficiently eliminate dysfunctional cells. The study of follicular apoptosis in small animals, especially in cats, is very important for the development of reproduction biotechnologies. Phenomenon of apoptosis showed no relationship with age in queens, occurring in a physiological, continuous and proportionate manner considering the number of nondominant follicles involved in each estrous cycle.

P–431 Human oocytes in-vitro maturation efficacy from infancy to adulthood – is there an optimal age?

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Karavani ◽  
P Wasserzug-Pash ◽  
T Mordechai-Daniel ◽  
M Klutstein ◽  
T Imbar
Keyword(s):  
Fertility Preservation ◽  
In Vitro Maturation ◽  
Ovarian Tissue ◽  
Age Groups ◽  
Ovarian Tissue Cryopreservation ◽  
Success Rates ◽  
Human Oocytes ◽  
Tertiary Center ◽  
Significant Difference

Abstract Study question Does human oocytes in-vitro maturation (IVM) effectiveness change throughout childhood, adolescence and adulthood in girls and women undergoing fertility preservation via ovarian tissue cryopreservation (OTC) prior to chemo-radiotherapy exposure? Summary answer The optimal age for IVM is from menarche to 25 years, while pre-menarche girls and women older than 30 years have extremely low maturation rates. What is known already In vitro maturation of oocytes from antral follicles seen during tissue harvesting is a fertility preservation technique with potential advantages over OTC, as mature frozen and later thawed oocyte used for fertilization poses decreased risk of malignant cells re-seeding, as compared to ovarian tissue implantation. We previously demonstrated that IVM performed following OTC in fertility preservation patients, even in pre-menarche girls, yields a fair amount of oocytes available for IVM and freezing for future use. Study design, size, duration A retrospective cohort study, evaluating IVM outcomes in chemotherapy naïve patients referred for fertility preservation by OTC that had oocyte collected from the medium with attempted IVM between 2003 and 2020 in a university affiliated tertiary center. Participants/materials, setting, methods A total of 133 chemotherapy naïve patients aged 1–35 years with attempted IVM were included in the study. The primary outcome was IVM rate in the different age groups – pre-menarche (1–5 years and ≥6 years), post-menarche (menarche–17 years), young adults (18–24 years) and adults (25–29 and 30–35 years). Comparison between paired groups for significant difference in the IVM rate parameter was done using the Tukey’s Studentized Range (HSD) Test. Main results and the role of chance A gradual increase in mean IVM rate was demonstrated in the age groups over 1 to 25 years (4.6% (1–5 years), 23.8% (6 years to menarche) and 28.4% (menarche to 17 years), with a peak of 38.3% in the 18–24 years group, followed by a decrease in the 25–29 years group (19.3%), down to a very low IVM rate (8.9%) in the 30–35 years group. A significant difference in IVM rates was noted between the age extremes – the very young (1–5 years) and the oldest (30–35 years) groups, as compared with the 18–24-year group (p &lt; 0.001). Number of oocytes matured, percent of patients with matured oocytes and overall maturation rate differed significantly (p &lt; 0.001). Limitations, reasons for caution Data regarding ovarian reserve evaluation was not available for most of the patients, due to our pre-op OTC procedures protocol. None of our patients have used their frozen in-vitro matured oocytes, as such further implications of age on in-vitro matured oocytes quality and implantation potential has yet to be evaluated. Wider implications of the findings: Our finding of extremely low success rates in those very young (under 6 years) and older (≥30 years) patients suggest that IVM of oocyte retrieved during OTC prior to chemotherapy should not be attempted in these age group. Trial registration number N/A

Adrenocortical function in aging exercise-trained rats

1977 ◽  
Vol 43 (5) ◽  
pp. 839-843 ◽  
Author(s):  
J. A. Severson ◽  
R. D. Fell ◽  
J. G. Tuig ◽  
D. R. Griffith
Keyword(s):  
Age Groups ◽  
Plasma Corticosterone ◽  
Treadmill Running ◽  
Adrenocortical Function ◽  
Elevated Plasma ◽  
Corticosterone Concentration ◽  
Relationship Of ◽  
The Relationship

Plasma corticosterone concentrations and in vitro adrenal secretion of corticosterone were determined in exercise-trained rats. Rats, 100, 200, and 300 days of age, were trained for a 10-wk period by treadmill running. Following the training program, rats were subjected to an acute bout of swimming. Acute swimming elevated plasma corticosterone concentrations in all age groups. At 170 days of age, the plasma corticosterone concentration following swimming was higher in exercise-trained rats than in controls. The opposite was true of acutely swum rats at 270 and 370 days of age. Acute swimming elevated the in vitro adrenal gland response to adrenocorticotropic hormone stimulation in control rats at all ages and in trained rats at 170 days of age. The in vivo relationship of epinephrine and the pituitary adrenal system is suggested as a mechanism which could have caused this response. The relationship of secretion rates to plasma corticosterone concentrations indicated that extra-adrenal mechanisms, such as decreased turnover, were also responsible for the elevated plasma corticosterone levels observed in response to acute swimming.

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 &lt; 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 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.

A selective increase in circulating inhibin and inhibin pro-αC at the time of ovulation in the mare

1999 ◽  
Vol 277 (5) ◽  
pp. E870-E875 ◽  
Author(s):  
Kentaro Nagaoka ◽  
Yasuo Nambo ◽  
Natsuko Nagamine ◽  
Shun-Ichi Nagata ◽  
Yumiko Tanaka ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Plasma Concentrations ◽  
Precursor Protein ◽  
Α Subunit ◽  
Ovulatory Follicles ◽  
The Relationship ◽  
Selective Increase ◽  
Estradiol 17Β

The relationship between a selective increase in circulating immunoreactive (ir)-inhibin and the time of ovulation was investigated in mares. Concentrations of plasma ir-inhibin were measured every 4 h during the periovulatory period. Inhibin pro-αC, a precursor protein of the inhibin α-subunit, was also measured. The changes in ir-inhibin and inhibin pro-αC in circulation were parallel. Concentrations of both ir-inhibin and inhibin pro-αC in the plasma increased at the same time when ovulatory follicles ruptured, and the peak levels of circulating ir-inhibin and inhibin pro-αC were maintained for 4–8 h. There was no selective increase in plasma concentrations of estradiol-17β during the process of ovulation. These results suggest that the selective increase in ir-inhibin and inhibin pro-αC was caused by the absorption of follicular fluid after the rupture of ovulatory follicles. These results also suggest that the measuring of plasma concentrations of ir-inhibin or inhibin pro-αC in mares might be a useful method for detecting the time of ovulation.

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.

scholarly journals The activin-follistatin system and in vitro early follicle development in goats

2006 ◽  
Vol 189 (1) ◽  
pp. 113-125 ◽  
Author(s):  
J R V Silva ◽  
T Tharasanit ◽  
M A M Taverne ◽  
G C van der Weijden ◽  
R R Santos ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Activin A ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Follicle Development ◽  
Cortical Tissue ◽  
Primary Follicle ◽  
Growth And Survival ◽  
Primordial Follicles

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 173-182 ◽  
Author(s):  
M.H.T. Matos ◽  
I.B. Lima-Verde ◽  
M.C.A. Luque ◽  
J.E. Maia Jr ◽  
J.R.V. Silva ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Follicle Stimulating Hormone ◽  
Control Medium ◽  
Primordial Follicles ◽  
Ultrastructural Studies ◽  
Follicle Diameter ◽  
Transmission Electron ◽  
Stimulating Hormone

SummaryThe aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM – control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.

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