Exploring the potential use of platelet rich plasma (PRP) from adult and umbilical cord blood in murine follicle culture.

Ovarian follicle culture is a powerful tool to study follicular physiology and has potential applications in clinical and commercial settings. Despite remarkable progress, recreating folliculogenesis in vitro remains challenging for many mammalian species. This study investigates the impact of platelet-rich plasma (PRP) derived from adult blood (human platelet lysate, hPL) and umbilical cord blood (Umbilical cord plasma, UCP) on murine pre-antral follicle culture and oocyte maturation. Pre-antral follicles were cultured individually for 10 days with fetal bovine serum (FBS) serving as the control and two PRP sources (hPL and UCP) and their activated forms (Ac-hPL and Ac-UCP). The results suggest that neither hPL nor UCP, regardless of activation status, improved follicle culture outcomes compared to FBS. Interestingly, activation did not significantly impact the main functional outcomes such as maturation rates, survival, and growth. Oestradiol secretion and oocyte diameter, often considered hallmarks of follicle quality, did not show significant differences between matured and non-matured oocytes across the treatment groups. However, gene expression analysis revealed a significant upregulation of Gdf-9 and Bmp-15 mRNA levels in oocytes from the Ac-UCP group, regardless of maturation stage, suggesting that the accumulation of the mRNA could be due to potential challenges in translation in the Ac-UCP group. In conclusion, this study challenges the hypothesis that PRP, as a serum source, could improve follicle culture outcomes compared to FBS, the gold standard in murine follicle culture. Further research is needed to understand the species-specific effects of PRP and explore other potential factors affecting follicle culture and oocyte quality.

Human platelet lysate improves the growth and survival of cultured human pre-antral follicles.

RESEARCH QUESTION: How do platelet-rich plasma products like human platelet lysate (HPL) and umbilical cord plasma (UCP) affect the growth and survival of isolated human pre-antral follicles in vitro? DESIGN: Human pre-antral follicles (n = 724; mean diameter: 75 µm; range: 46-237 µm) were isolated from ovarian medulla donated by 14 patients undergoing unilateral oophorectomy for ovarian tissue cryopreservation. Follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with 5% fetal bovine serum (FBS) (n = 171), 2.5% human serum albumin (HSA) (n = 159), 5% HPL (n = 223) or 5% UCP (n = 171). RESULTS: The survival probability was significantly higher in the group supplemented with HPL (80%) compared with the other three groups: FBS (54%, P < 0.001); HSA (63%, P = 0.004) and UCP (29%, P < 0.001). Surviving follicles in the UCP group had less defined follicular membranes and decompacted granulosa cell layers. The median growth of surviving follicles was significantly (P < 0.001) larger in the HPL group (73 µm) compared with any of the other three groups: HSA (43 µm); FBS (40 µm) UCP (54 µm). A descriptive analysis of follicular secretion of anti-Müllerian hormone and oestradiol did not reveal any difference between the groups. The detectability of follicular genes was high for AR (100%), AMHR2 (100%) and FSHR (76%), whereas few follicles expressed LHR (20%). CONCLUSION: Human platelet lysate significantly improved survival and growth of cultured human pre-antral follicles compared with FBS, HSA and UCP. The use of HPL is a valuable improvement to culture human pre-antral follicles but further studies will have to prove whether the superiority of HPL translates into better quality oocytes.

Vitrification of canine ovarian tissue using the Ovarian Tissue Cryosystem (OTC) device.

The present study evaluated the effect of Ovarian Tissue Cryosystem (OTC) on follicular morphology and density, as well as on stromal cell density of vitrified canine ovarian tissue. Canine ovarian fragments collected from adult female dogs in stages of the random oestrous cycle were fixed (FC, fresh control) or vitrified (VIT) with an OTC device. After vitrification and warming, the fragments were fixed for histological analysis. Overall, the mean percentage of normal pre-antral follicles decreased after vitrification procedure (FC: 74.5% ± 1.6% vs. VIT: 52.05% ± 1.5%). Although the rates of normal primordial (71.1% ± 1.8%) and secondary (0.7% ± 0.4%) follicles vitrified showed a reduction (p < .05), vitrification using OTC showed considerable preservation of follicles, when compared to the fresh control (81.1% ± 1.5% and 2.3% ± 0.6%, respectively). The mean follicular density was maintained after vitrification (FC: 199.65 ± 12.8 vs. VIT: 199.68 ± 10.8), whereas the stromal cell density decreased in the VIT group. Based on the results, we recommend the use of OTC for vitrification of canine ovarian tissue.

Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification.

PURPOSE: Gap junctions and transzonal projections play a crucial role in intercellular communication between different follicular components and are necessary for follicle development. We aimed to demonstrate gap junction protein connexin 43 (Cx43) and transzonal projections (TZPs) in viable, category 1, isolated bovine pre-antral follicles (PAFs) during short-term culture and after vitrification and warming. METHODS: This study involved four experimental groups: fresh control, 2-day culture, 4-day culture, and vitrified secondary PAFs. Isolated PAFs were vitrified using a simple and efficient cryopreservation method by means of mini cell strainers. RESULTS: Cx43 and TZPs were detected in pre-antral follicles of all stages, as well as in every experimental group. The group fresh follicles showed a higher percentage of follicles that were positive for Cx43 (91.7%) than the follicles that were vitrified (77.4%). All follicles that were cultured for 2 days were Cx43-positive (100%). Follicles cultured for 4 days (65.8%) (P = 0.002) showed the lowest percentage of follicles that were Cx43-positive. The percentages of the presence or (partial) absence of the TZP network were shown to be very heterogeneous between follicles in different treatment groups. CONCLUSIONS: These results suggest the maintenance of communication between the oocyte and the somatic companion cells after vitrification and warming. The varying percentages of the expression of the TZP network within groups suggests that it will be of interest to investigate whether this is truly due to variability in TZP integrity and follicle quality or due to methodological limitations.

Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold.

STUDY QUESTION: Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? SUMMARY ANSWER: We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. WHAT IS KNOWN ALREADY: Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. STUDY DESIGN, SIZE, DURATION: Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24 hours followed by 24 hours of 1 mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N = 241) and human (N = 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Incubation in 0.1% SDS for 18-24 hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. STUDY FUNDING/COMPETING INTEREST(S): This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

Effects of vitrification on the viability of alginate encapsulated isolated bovine pre-antral follicles.

PURPOSE: Individual follicle cryopreservation techniques, without hydrogel support, are labor-intensive and a substantial proportion of isolated follicles are lost during handling and after warming. Therefore, the viability and morphology of isolated bovine (as a model for human) pre-antral follicles after vitrification and warming, when encapsulated in alginate beads, were investigated. METHODS: Bovine pre-antral follicles were mechanically isolated and divided into four different groups: (1) culture in 2% alginate beads (3D system) and vitrification in beads using mesh cups (3DVIT), (2) culture in 2% alginate beads (3DCUL), (3) culture in 96-well plates (2D system) and vitrification using High Security Vitrification straws® (2DVIT), (4) culture in a 2D system (2DCUL). The same vitrification and warming protocols were used for embedded (3DVIT) and non-embedded follicles (2DVIT). RESULTS: No differences were observed in follicle viability between group 2DCUL and 3DCUL. Group 3DVIT showed the lowest viability (45.9%) according to calcein and neutral red staining among all groups. Group 2DVIT displayed the highest viability (87.5%) and largest percentage of follicles with a well-preserved morphology. CONCLUSIONS: Our results show that, using a vitification protocol optimized for non-embedded follicles, 2D culture is more effective in vitrifying isolated follicles. However, embedding in alginate allow to handle follicles more efficiently, i.e., without excessive manipulation and thus less labor-intensive in combination with a reduced loss of follicles during the procedure. Based on the increased work efficiency, but lower viability and higher proportion of follicles showing impaired morphology, we consider it advantageous to optimize the protocol for the vitrification of embedded follicles to increase survival and maintain morphology after vitrification.

The development and integrity of equine pre-antral follicles cultured in vitro with follicle-stimulating hormone (FSH) supplementation.

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre-antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre-antral follicles, the groups with 100 ng/ml FSH of 2-days culture as well as 50, 100 and 200 ng/ml FSH of 6-days culture provided the best results. In conclusion, the in vitro culture of abattoir-derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.

Determination of the normal reference interval for anti-Müllerian hormone (AMH) in bitches and use of AMH as a potential predictor of litter size.

Anti-Müllerian hormone (AMH) is a reliable endocrine marker of ovarian reserve in many species with extensive literature in both humans and cattle. However, there are no known hormonal predictors of ovarian reserve and potential reproductive performance in the bitch. A prospective cohort study was performed involving 155 intact bitches of various ages (range 1.2-7.6 years) and breeds that were presented for routine breeding management over a one-year period. All bitches were artificially inseminated with frozen or fresh semen using the transcervical insemination (TCI) technique. AMH concentrations were measured using a commercially available canine AMH ELISA (Ansh Labs® , Texas, USA), which we validated prior to performing the study. The reference interval (RI) for AMH for all bitches in the study, regardless of body weight, was 2.9-21.1 ng/ml. There was a significant effect of bitch size and age on AMH concentrations. The RI for giant breeds was significantly (p < .01) lower (1.75-15.6 ng/ml) than small-sized (5.6-24.2 ng/ml), medium-sized (4.3-23.7 ng/ml) and large-sized (4.3-21.0 ng/ml) bitches. The mean AMH concentration in bitches less than 4 years of age was 12.4 ng/ml, whereas the mean AMH concentration in bitches older than 4 years of age was 10.5 ng/ml (p < .05). For each additional year of age above 1 year of age, AMH concentrations fell by 0.5 ng/ml. There was no effect of AMH concentration on the whelping rate. Smaller breeds had smaller litters (and higher AMH concentrations), but within each breed size category, bitches with higher AMH concentrations had significantly larger litter sizes (p < .01). For each 1 ng/ml increase in AMH, litter size increased by 0.3 pups/litter. In conclusion, we determined a normal reference interval for AMH in bitches based on body weight using a canine-specific assay. In agreement with findings in humans and other species, we found that there is a decline in AMH concentrations with advancing age in bitches. Finally, the significant positive correlation between AMH concentrations and litter size indicates that AMH may be a useful management tool for the selection of bitches in breeding programmes.

Vitrification affects the expression of matrix metalloproteinases and their tissue inhibitors of mouse ovarian tissue.

BACKGROUND: One of the most major obstacles of ovarian tissue vitrification is suboptimal developmental competence of follicles. Matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) and their tissue inhibitors TIMP-1 and TIMP-2 are involved in the remodeling of the extracellular matrix in the ovaries. OBJECTIVE: This study aimed to evaluate the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 genes in the preantral follicles derived from vitrified mouse ovaries. MATERIALS AND METHODS: In this experimental study, the gene expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the isolated preantral follicles derived from fresh and vitrified ovaries of 14-16 days old female mice through real time qRT-PCR was evaluated. Developmental parameters, including survival rate, growth, antrum formation and metaphase II oocytes were also analyzed. RESULTS: The developmental parameters of fresh preantral follicles were significantly higher than vitrified preantral follicles. The TIMP-1 and MMP-9 expression levels showed no differences between fresh and vitrified preantral follicles (p=0.22, p=0.11 respectively). By contrast, TIMP-2 expression significantly decreased (p=0.00) and MMP-2 expression increased significantly (p=0.00) in vitrified preantral follicles compared with to fresh ones. CONCLUSION: Changes in expression of MMP-2 and TIMP-2 after ovarian tissues vitrification is partially correlated with decrease in follicle development.

Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.

STUDY QUESTION: Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? SUMMARY ANSWER: Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. WHAT IS KNOWN ALREADY: Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. STUDY DESIGN, SIZE, DURATION: A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. MAIN RESULTS AND ROLE OF CHANCE: Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group). LIMITATIONS, REASONS FOR CAUTION: There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step. WIDER IMPLICATIONS OF THE FINDINGS: Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy. STUDY FUNDING/COMPETING INTERESTS: The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests. TRIAL REGISTRATION NUMBER: N/A.

Influence of follicle stage on artificial ovary outcome using fibrin as a matrix.

STUDY QUESTION: Do primordial-primary versus secondary follicles embedded inside a fibrin matrix have different capabilities to survive and grow after isolation and transplantation? SUMMARY ANSWER: Mouse primordial-primary follicles showed a lower recovery rate than secondary follicles, but both were able to grow. WHAT IS KNOWN ALREADY: Fresh isolated mouse follicles and ovarian stromal cells embedded in a fibrin matrix are capable of surviving and developing after short-term autografting. STUDY DESIGN, SIZE, DURATION: In vivo experimental model using 11 donor Naval Medical Research Institute (NMRI) mice and 11 recipient severe combined immunodeficiency (SCID) mice. Both ovaries from all NMRI mice were mechanically disrupted and primordial-primary and secondary follicles were isolated with ovarian stromal cells. They were then encapsulated in a fibrin matrix composed of 12.5 mg/ml of fibrinogen (F12.5) and 1 IU/ml of thrombin (T1) (F12.5/T1), and grafted to the inner part of the peritoneum of SCID mice for 2 and 7 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to conduct histological (H-E staining) and immunohistochemical (Ki67, TUNEL) analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Although all grafted fibrin clots were recovered, the follicle recovery rate on day 2 was 16 and 40% for primordial-primary and secondary follicles respectively, while on day 7, it was 6 and 28%. The secondary group showed a significantly higher recovery rate than the primordial-primary group (23%, P-value <0.001). Follicles found in both groups were viable, as demonstrated by live/dead assays, and no difference was observed in the apoptosis rate between groups, as evidenced by TUNEL. Their growth to further stages was confirmed by Ki67 immunostaining. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results, secondary follicles appear to be more likely to survive and develop than primordial-primary follicles in a fibrin matrix after both periods of grafting. These findings may also be attributed to the specific features of the fibrin matrix, which could benefit larger follicles, but not smaller follicles. WIDER IMPLICATIONS OF THE FINDINGS: This study is essential to understanding possible impairment caused by factors such as the isolation procedure or fibrin matrix composition to the survival and development of different follicle stages. It therefore provides the basis for further investigations with longer periods of grafting. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4578.14 and 7.4627.13, grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, the Foundation Against Cancer, and the Region Wallone (Convention N°6519-OVART) and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any competing interests to declare.

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

This 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.

Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue.

STUDY QUESTION: Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue? SUMMARY ANSWER: Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol. WHAT IS KNOWN ALREADY: Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. The influence of the vitrification procedure on the cooling rate was evaluated by a computer model. MAIN RESULTS: After vitrification, warming and long-term grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Müllerian hormone (AMH) and growth differentiation factor-9 (GDF-9) immunostaining. Corpora lutea were also observed, evidencing successful ovulation in all the animals. Stromal tissue quality did not appear to be negatively affected by our cryopreservation procedure, as demonstrated by vascularization and proportions of fibrotic areas, which were similar to those found in fresh ungrafted ovarian tissue. LIMITATIONS, REASONS FOR CAUTION: Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies. WIDER IMPLICATIONS OF THE FINDINGS: In addition to encouraging results obtained with our vitrification procedure for non-human ovarian tissue, this study also showed, for the first time, expression of AMH and GDF-9 in ovarian follicles. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4507.10, grant 3.4.590.08 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, and Department of Mechanical Engineering at Louisiana State University (support to Ram Devireddy), and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors has any competing interests to declare.

Goat ovarian follicles express different levels of mRNA for inhibin-ßA subunit and activin-A stimulates secondary follicle growth in vitro / Folículos ovarianos caprinos expressam diferentes níveis de RNAm para subunidade ßA da inibina e ativina-A promove o crescimento de folículos secundários in vitro

This study investigated the levels of messenger ribonucleic acids (mRNA) for inhibin-ßA subunit in goat primordial, primary and secondary follicles, as well as in cumulus-oocyte complexes (COCs) and mural granulosa / theca cells of antral follicles. The effects of activin-A (100ng mL-1) and/or follicle stimulating hormone (FSH, 50ng mL-1) on growth and expression of mRNA for activin-A and FSH receptor (FSH-R) in secondary follicles cultured for six days were evaluated. The data showed that the expression of inhibin-ßA is lower in secondary follicles than in primary follicles and is higher in large antral follicles than in small antral follicles. After culture, activin-A and/or FSH promoted growth of secondary follicles, while FSH increased the levels of mRNA for inhibin-ßA, and activin-A increased the levels of FSH-R mRNA. In conclusion, mRNA for inhibin-ßA is expressed at different levels in pre-antral and antral follicles and activin-A acts as a stimulator of the FSH-R expression in goat follicles. On its turn, the expression of inhibin-ßA is stimulated by FSH, which together with activin-A promotes secondary follicle growth in-vitro.

Este estudo investigou os níveis de ácidos ribonucleicos (RNAm) para a subunidade ßA da inibina em folículos primordiais, primários e secundários caprinos, bem como em complexos cumulus-oócitos (CCOs) e células da granulosa mural/teca de folículos antrais. Além disso, avaliaram-se os efeitos da ativina-A (100ng mL-1) e/ou hormônio folículo estimulante (FSH, 50ng mL-1) sobre o crescimento e a expressão do RNAm para inibina-ßA e receptores de FSH (FSH-R) em folículos secundários cultivados por seis dias. Os dados mostraram que a expressão de inibina-ßA é menor em folículos secundários do que em folículos primários e é maior em grandes folículos antrais que nos pequenos folículos antrais. Após o cultivo, ativina-A e/ou FSH promoveram o crescimento de folículos secundários. Enquanto o FSH aumentou os níveis de RNAm para inibina-ßA, a ativina-A aumentou os níveis de RNAm para FSH-R. Em conclusão, a inibina-ßA é expressa em diferentes níveis em folículos pré-antrais e antrais e a ativina-A atua como um estimulador da expressão de FSH-R em folículos caprinos. Por sua vez, a expressão de inibina-ßA é estimulada pelo FSH, que, juntamente com ativina, promove o crescimento de folículos secundários in vitro.

Ovarian folliculogenesis in collared peccary: Pecari tajacu (Artiodactyla: Tayassuidae)

The sustainability and production of collared peccary (Pecari tajacu) has been studied in the last few years; however, further information on its reproduction is necessary for breeding systems success. Understanding folliculogenesis aspects will contribute to effective reproductive biotechniques, which are useful in the preservation and production of wildlife. The aim of this study was to evaluate the ovarian folliculogenesis in collared peccary. Ovaries from six adult females of collared peccary were obtained through ovariectomy and analyzed. These were fixed in aqueous Bouin’s solution and sectioned into 7μm slices, stained with hematoxilin-eosin and analyzed by light microscopy. The number of pre-antral and antral follicles per ovary was estimated using the Fractionator Method. The follicles, oocytes and oocyte nuclei were measured using an ocular micrometer. Results showed that the length, width, thickness, weight, and the gross anatomy of the right and left ovaries were not significantly different. However, the mean number of corpora lutea was different between the phases of the estrous cycle (p<0.05), with the highest mean in the luteal phase. Primordial follicles were found in the cortex; the oocytes were enveloped by a single layer of flattened follicular cells. In the primary follicles, proliferation of the follicular cells gave rise to cuboidal cells (granulosa cells). The secondary follicle was characterized by two or more concentric layers of cuboidal cells (granulosa), beginning of antrum formation, and the presence of pellucid zone and theca cells. Antral follicles were characterized by a central cavity (antrum), the presence of cumulus oophorus and theca layers (interna and externa). In the right ovary, the values of the primordial and primary follicles were similar, but significantly different from the secondary ones (p<0.05). In the left ovary, significant differences were observed between all follicles in the follicular phase (p<0.05); the mean number of primordial and primary follicles was similar in the luteal phase. The mean number of pre-antral follicles and antral follicles in the follicular phase was higher in the left ovary (p<0.05). The mean number of antral follicles in the luteal phase was similar in both ovaries. We also found significant differences in mean diameter of preantral follicles, oocyte, granulosa layer and oocyte nucleus during the estrous cycle. In the antral follicles a significant difference was observed only in follicular diameter (p<0.05). The predominance of active primordial and primary follicles was found in both phases; otherwise the secondary follicles and antral follicles showed a high degree of degeneration. The results obtained in the present work will strengthen the development of biotechnology programs to improve the productive potential and conservation of the collared peccary.

La sustentabilidad y la producción de pecarí de collar (Pecari tajacu) han sido estudiados en los últimos años, sin embargo, más información sobre su reproducción es necesaria para el éxito de los sistemas de crianza . La comprensión de los aspectos relacionados con la foliculogénesis contribuirá con la aplicación de biotécnicas de reproducción, las cuales son útiles en la preservación y la producción de la vida silvestre. El objetivo de este estudio fue obtener datos sobre la población folicular del ovario de pecarí de collar. En relación con la población folicular en el ovario derecho, los valores de los folículos primordiales y primarios fueron similares, pero se observó que había una diferencia significativa (p<0.05) con el secundario. En el ovario izquierdo, la fase folicular presentó diferencias significativas (p<0.05) entre todos los folículos, y en la fase lútea el número medio de folículos primordiales y primarios fueron similares. Ahora bien, con respecto a la población de folículos antrales, en la fase folicular, se observaron diferencias significativas entre los ovarios (p<0.05), y de forma similar en la fase lútea. También se encontraron diferencias significativas en el diámetro medio de los folículos preantrales, oocitos, la capa granulosa y el núcleo del oocito durante las fases del ciclo estral. Asimismo, en los folículos antrales se observó diferencia estadísticamente significativa (p<0.05) en el diámetro folicular. En ambas fases del ciclo estral, se encontró el predominio de folículos primordiales y primarios en desarrollo, por otro lado, los folículos secundarios y los folículos antrales mostraron un alto grado de degeneración. Los resultados aquí presentes son necesarios para el desarrollo de los programas de mejoramiento y conservación.

Establishment of Culture System for In Vitro Culture of Mouse Pre-antral Follicles: Effect on In Vitro Growth by Thecal-Stromal Cells in Culture Medium without Hormones / 대한산부인과학회잡지

OBJECTIVE: This present study was conducted to examine the effects on in vitro growth of pre-antral mouse follicles by thecal-stromal cells attached to around the follicular membrane in culture medium without hormones. METHODS: Pre-antral follicles (100-130 micro meter) used in our studies were isolated mechanically by fine 30 G needles attached to 1 ml insulin syringe from mice ovaries of 20-25 days old female ICR strain. Isolated pre-antral follicles were divided into three groups by attached status of thecal-stromal cell layers to follicular membrane. Follicular Initial diameters of group I was 112.5 (mean) +/- 6.2 (SD) micro meter (n=31), group II was 112.8 +/- 7.8 micro meter (n=23), and group III was 115.1 +/- 6.5 micro meter (n=27). Divided pre-antral follicles were washed in fresh Ham's F-10 medium and cultured in 20 micro liter droplets of DMEM with glutamine, glucose and pyruvate under mineral oil on the 60 mm culture dish. All experimental media were supplemented with 10% FBS without hormones. Media were exchanged wholly by fresh media every 2 days in culture. Diameters of intrafollicular oocytes and cultured pre-antral follicles were measured using an precalibrated cross ocular micrometer at X200 every day during 6 days in vitro culture. Results were considered statistically significant when p value was less than 0.05 using Student's t-test. RESULTS: The mean and standard deviation (SD) of initial diameters of pre-antral follicles was 113.5 +/- 2.2 micro meter in three groups. Follicular growth rate of group III was significantly (p<0.05) higher in whole culture periods than group I and group II. Rate of follicular growth between group I and group II were not significant difference in culture for 1 to 3 days. However, group II was significantly higher than group I in culture for 4 days. Diameters of grown follicles for 6 days was 155.2 +/- 18.7 micro meter, 196.9 +/- 24.1 micro meter and 284.2 +/- 47.6 micro meter in group I, group II and group III, respectively. Growth rate of intrafollicular oocytes between three groups were not difference and revealed to continuous growth patterns in whole culture periods. Follicular diameter after culture for 7 days were not measured because of disruption of follicular structure or outgrowth of granulosa cells. CONCLUSION: Pre-antral follicles were grown very well in DMEM medium without hormones. Thecal- stromal cells attached to the surface of follicular membrane has acted effectively in vitro growth of follicles.