Translational Physiology of Anti-Müllerian Hormone: Clinical Applications in Female Fertility Preservation and Cancer Treatment

BackgroundWhilst the ability of AMH to induce the regression of the Müllerian ducts in the male fetus is well appreciated, AMH has additional biological actions in relation to steroid biosynthesis and ovarian follicle dynamics. An understanding of the physiology of AMH illuminates the potential therapeutic utility of AMH to protect the ovarian reserve during chemotherapy and in the treatment of female malignancies. The translation of the biological actions of AMH into clinical applications is an emerging focus of research, with promising preliminary results.Objective and RationaleStudies indicate AMH restrains primordial follicle development, thus administration of AMH during chemotherapy may protect the ovarian reserve by preventing the mass activation of primordial follicles. As AMH induces regression of tissues expressing the AMH receptor (AMHRII), administration of AMH may inhibit growth of malignancies expressing AMHR II. This review evaluates the biological actions of AMH in females and appraises human clinical applications.Search MethodsA comprehensive search of the Medline and EMBASE databases seeking articles related to the physiological functions and therapeutic applications of AMH was conducted in July 2021. The search was limited to studies published in English.OutcomesAMH regulates primordial follicle recruitment and moderates sex steroid production through the inhibition of transcription of enzymes in the steroid biosynthetic pathway, primarily aromatase and 17α-hydroxylase/17,20-lyase. Preliminary data indicates that administration of AMH to mice during chemotherapy conveys a degree of protection to the ovarian reserve. Administration of AMH at the time of ovarian tissue grafting has the potential to restrain uncontrolled primordial follicle growth during revascularization. Numerous studies demonstrate AMH induced regression of AMHR II expressing malignancies. As this action occurs via a different mechanism to traditional chemotherapeutic agents, AMH has the capacity to inhibit proliferation of chemo-resistant ovarian cancer cells and cancer stem cells.Wider ImplicationsTo date, AMH has not been administered to humans. Data identified in this review suggests administration of AMH would be safe and well tolerated. Administration of AMH during chemotherapy may provide a synchronistic benefit to women with an AMHR II expressing malignancy, protecting the ovarian reserve whilst the cancer is treated by dual mechanisms.

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Impact of nicotinamide mononucleotide on transplanted mouse ovarian tissue

Reproduction ◽

10.1530/rep-20-0539 ◽

2020 ◽

Author(s):

Michael J Bertoldo ◽

Valentina Rodriguez Paris ◽

Debra A Gook ◽

Melissa C Edwards ◽

Katherine Wu ◽

...

Keyword(s):

Ovarian Tissue ◽

Ovarian Follicle ◽

Primordial Follicle ◽

Young Female ◽

Female Adolescent ◽

Ovarian Tissue Cryopreservation ◽

Post Transplantation ◽

Primordial Follicles ◽

Nicotinamide Mononucleotide ◽

The Impact

Ovarian tissue cryopreservation and future transplantation is the only strategy to preserve the fertility of young female adolescent and pre-pubertal patients. The primary challenge to ovarian graft longevity is the substantial loss of primordial follicles during the period of ischemia post-transplantation. Nicotinamide mononucleotide (NMN), a precursor of the essential metabolite nicotinamide adenine dinucleotide (NAD+), is known to reduce ischemic damage. Therefore, the objective of the current study was to assess the impact of short- and long-term NMN administration on follicle number and health following ovarian tissue transplantation. Hemi-ovaries from C57Bl6 mice (n=8-12/group) were transplanted under the kidney capsule of bilaterally ovariectomised severe combined immunodeficient (SCID) mice. Recipient mice were administered either normal drinking water or water supplemented with NMN (2g/L) for either 14 or 56 days. At the end of each treatment period ovarian transplants were collected. There was no effect of NMN on the resumption of oestrous or length of oestrous cycles. Transplantation significantly reduced the total number of follicles with the greatest impact observed at the primordial follicle stage. We report that NMN did not prevent this loss. While NMN did not significantly impact the proportion of apoptotic follicles, NMN normalised PCNA expression at the primordial and intermediate stages but not at later stages. In conclusion, NMN administration did not prevent ovarian follicle loss under the conditions of this study.

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Unraveling the mechanisms of chemotherapy-induced damage to human primordial follicle reserve: road to developing therapeutics for fertility preservation and reversing ovarian aging

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa043 ◽

2020 ◽

Vol 26 (8) ◽

pp. 553-566 ◽

Cited By ~ 4

Author(s):

Katarzyna J Szymanska ◽

Xiujuan Tan ◽

Kutluk Oktay

Keyword(s):

Ovarian Reserve ◽

Primordial Follicle ◽

Chemotherapeutic Agents ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Microvascular Damage ◽

Primordial Follicles ◽

Strand Breaks ◽

Apoptotic Death ◽

Dna Dsbs

Abstract Among the investigated mechanisms of chemotherapy-induced damage to human primordial follicle reserve are induction of DNA double-strand breaks (DSBs) and resultant apoptotic death, stromal–microvascular damage and follicle activation. Accumulating basic and translational evidence suggests that acute exposure to gonadotoxic chemotherapeutics, such as cyclophosphamide or doxorubicin, induces DNA DSBs and triggers apoptotic death of primordial follicle oocytes within 12–24 h, resulting in the massive loss of ovarian reserve. Evidence also indicates that chemotherapeutic agents can cause microvascular and stromal damage, induce hypoxia and indirectly affect ovarian reserve. While it is possible that the acute reduction of the primordial follicle reserve by massive apoptotic losses may result in delayed activation of some primordial follicles, this is unlikely to be a predominant mechanism of loss in humans. Here, we review these mechanisms of chemotherapy-induced ovarian reserve depletion and the potential reasons for the discrepancies among the studies. Based on the current literature, we propose an integrated hypothesis that explains both the acute and delayed chemotherapy-induced loss of primordial follicle reserve in the human ovary.

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Distinct Signaling Pathways Distinguish in vivo From in vitro Growth in Murine Ovarian Follicle Activation and Maturation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.708076 ◽

2021 ◽

Vol 9 ◽

Author(s):

Mahboobeh Amoushahi ◽

Karin Lykke-Hartmann

Keyword(s):

Ovarian Tissue ◽

Ovarian Follicle ◽

Primordial Follicle ◽

Ovarian Tissue Cryopreservation ◽

Ros Production ◽

Primordial Follicles ◽

Expression Of Genes ◽

Follicle Activation

Women with cancer and low ovarian reserves face serious challenges in infertility treatment. Ovarian tissue cryopreservation is currently used for such patients to preserve fertility. One major challenge is the activation of dormant ovarian follicles, which is hampered by our limited biological understanding of molecular determinants that activate dormant follicles and help maintain healthy follicles during growth. Here, we investigated the transcriptomes of oocytes isolated from dormant (primordial) and activated (primary) follicles under in vivo and in vitro conditions. We compared the biological relevance of the initial molecular markers of mature metaphase II (MII) oocytes developed in vivo or in vitro. The expression levels of genes involved in the cell cycle, signal transduction, and Wnt signaling were highly enriched in oocytes from primary follicles and MII oocytes. Interestingly, we detected strong downregulation of the expression of genes involved in mitochondrial and reactive oxygen species (ROS) production in oocytes from primordial follicles, in contrast to oocytes from primary follicles and MII oocytes. Our results showed a dynamic pattern in mitochondrial and ROS production-related genes, emphasizing their important role(s) in primordial follicle activation and oocyte maturation. The transcriptome of MII oocytes showed a major divergence from that of oocytes of primordial and primary follicles.

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Single-Oocyte Gene Expression Suggests That Curcumin Can Protect the Ovarian Reserve by Regulating the PTEN-AKT-FOXO3a Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22126570 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6570

Author(s):

Yue Lv ◽

Rui-Can Cao ◽

Hong-Bin Liu ◽

Xian-Wei Su ◽

Gang Lu ◽

...

Keyword(s):

Gene Expression ◽

Ovarian Reserve ◽

Primordial Follicle ◽

Term Treatment ◽

New Drugs ◽

Gene Profiling ◽

Primordial Follicles ◽

Long Term Treatment ◽

Follicle Activation

A better understanding of the mechanism of primordial follicle activation will help us better understand the causes of premature ovarian insufficiency (POI), and will help us identify new drugs that can be applied to the clinical treatment of infertility. In this study, single oocytes were isolated from primordial and primary follicles, and were used for gene profiling with TaqMan array cards. Bioinformatics analysis was performed on the gene expression data, and Ingenuity Pathway Analysis was used to analyze and predict drugs that affect follicle activation. An ovarian in vitro culture system was used to verify the function of the drug candidates, and we found that curcumin maintains the ovarian reserve. Long-term treatment with 100 mg/kg curcumin improved the ovarian reserve indicators of AMH, FSH, and estradiol in aging mice. Mechanistic studies show that curcumin can affect the translocation of FOXO3, thereby inhibiting the PTEN-AKT-FOXO3a pathway and protecting primordial follicles from overactivation. These results suggest that curcumin is a potential drug for the treatment of POI patients and for fertility preservation.

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

Human Reproduction Open ◽

10.1093/hropen/hoaa048 ◽

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.

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In vitro follicle culture in the context of IVF

Reproduction ◽

10.1530/rep-18-0173 ◽

2018 ◽

Vol 156 (1) ◽

pp. F59-F73 ◽

Cited By ~ 10

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.

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

Human Reproduction ◽

10.1093/humrep/deab130.448 ◽

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

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138 Relationships Between Antral Follicle Count, Ovarian Volume, Pre-Antral Follicle Number, and Oocyte Quality

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab138 ◽

2018 ◽

Vol 30 (1) ◽

pp. 209

Author(s):

G. L. Vasconcelos ◽

R. Maculan ◽

N. Alves ◽

A. L. A. P. L. Ribeiro ◽

A. W. B. Silva ◽

...

Keyword(s):

Ovarian Follicle ◽

Primordial Follicle ◽

Antral Follicle ◽

Oocyte Quality ◽

Antral Follicle Count ◽

Primary Follicle ◽

Ovarian Volume ◽

Primordial Follicles ◽

Antral Follicles ◽

Grade 3

The objective was to evaluate the possible relationships between AFC, ovarian volume, ovarian follicle reserve and oocyte quality in abattoir-derived ovaries (experiment 1) and in cows (experiment 2) submitted to OPU. Antral follicle counts of ≥25, 16 to 24, and ≤ 16 were used to define AFC classes as high (HAFC), intermediate (IAFC), and low (LAFC) in both experiments. In experiment 1, after antral follicles were aspirated, abattoir ovaries (n = 10 per AFC class) were processed by conventional histology and pre-antral follicles were counted within primordial, primary, secondary, and tertiary classes and classified as either healthy or degenerate under regular microscopy (Cushman et al. 1999). In experiment 2, HAFC (n = 42), IAFC (n = 34), and LAFC (n = 29) cows were submitted to OPU and oocytes classified as grades 1, 2, and 3 or degenerate (IETS, 2010). Antral follicles (≥3 mm in diameter) were counted by ultrasonography. Data were analysed by GENMOD and GLM procedures of SAS (SAS Institute Inc., Cary, NC, USA) after transformations, when required. In experiment 1, mean normal primordial follicle number was higher (P < 0.001) in HAFC (137.0 ± 1.6)a compared with IAFC (52.6 ± 1.9)b and LAFC (20.2 ± 5.3)c ovaries. However, the mean number of degenerate primordial follicles was lower (P < 0.001) in low count ovaries (2.4 ± 0.6) compared with HAFC (19.0 ± 4.7) and IAFC (16.4 ± 1.5, P < 0.001). Normal primary follicle number was higher in the HAFC compared with IAFC and LAFC ovarian classes (86.2 ± 7.0a v. 34.6 ± 5.1b and 14.4 ± 3.3c, respectively; P < 0.01). Degenerate primary follicles were higher in the HAFC compared with LAFC ovarian class (16.8 ± 6.5 v. 5.2 ± 2.64; P < 0.05). Normal secondary follicle number was also higher in the HAFC compared to LAFC ovarian classes (25.2 ± 7.67 v. 2.4 ± 0.8; P < 0.05). The number of degenerate secondary follicles differed (P < 0.01) only between the IAFC and the LAFC ovarian classes (0.6 ± 0.4 and 7.2 ± 2.4, respectively), which were similar (P > 0.5) to the HAFC class (3.8 ± 1.0). In experiment 2, grade 1, 2, and 3 oocytes, viable oocytes, and ovarian volume (mm3) were higher (P < 0.001) in HAFC compared with IAFC and LAFC cows (grade 1: 7.9 ± 0.6a, 4.9 ± 0.7b and 3.3 ± 0.7c; grade 2: 4.0 ± 0.4a, 2.8 ± 0.4b and 1.2c; grade 3: 2.1 ± 0.4a, 2.5 ± 0.4a and 1.3 ± 0.5b, respectively; viable oocytes: 16.3 ± 1.1a, 13.1 ± 1.2b, and 8.1 ± 1.3c, respectively; (volumes: 12.6 ± 0.7a, 10.1 ± 0.8b, and 8.1 ± 0.9c, respectively). In conclusion, high AFC is linked to a higher follicular reserve, oocyte quality, and ovarian volume. It is safe to apply AFC in the selection of bovine females without compromising oocyte or pre-antral follicular population qualities.

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

Zygote ◽

10.1017/s0967199414000173 ◽

2014 ◽

Vol 23 (4) ◽

pp. 537-549 ◽

Cited By ~ 3

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

Journal of Endocrinology ◽

10.1677/joe.1.06487 ◽

2006 ◽

Vol 189 (1) ◽

pp. 113-125 ◽

Cited By ~ 35

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.

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