Stroma cell-derived factor 1 and connexins (37 and 43) are preserved after vitrification and in vitro culture of goat 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.

Download Full-text

In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women

Human Reproduction ◽

10.1093/humrep/deaa246 ◽

2020 ◽

Vol 35 (12) ◽

pp. 2793-2807

Author(s):

P Asiabi ◽

M M Dolmans ◽

J Ambroise ◽

A Camboni ◽

C A Amorim

Keyword(s):

Growth Factors ◽

In Vitro Culture ◽

Protein Detection ◽

Basic Medium ◽

Specific Marker ◽

Mrna Levels ◽

Theca Cells ◽

Ovarian Cortex ◽

Steroid Dehydrogenase

Abstract STUDY QUESTION Can human theca cells (TCs) be differentiated in vitro? SUMMARY ANSWER It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones. WHAT IS KNOWN ALREADY There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs. STUDY DESIGN, SIZE, DURATION After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53–74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line. PARTICIPANTS/MATERIALS, SETTING, METHODS To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Results obtained from qPCR showed a significant increase (P < 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P < 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P < 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q < 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q < 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells. WIDER IMPLICATIONS OF THE FINDINGS This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests.

Download Full-text

Effects of α-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/s0102-09352009000100009 ◽

2009 ◽

Vol 61 (1) ◽

pp. 57-65 ◽

Cited By ~ 8

Author(s):

I.B. Lima-Verde ◽

M.H.T. Matos ◽

J.B. Bruno ◽

F.S. Martins ◽

R.R. Santos ◽

...

Keyword(s):

In Vitro Culture ◽

Ovarian Tissue ◽

Control Culture ◽

Ultrastructural Analysis ◽

Minimum Essential Medium ◽

Preantral Follicles ◽

Ovarian Cortex

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2% and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

Download Full-text

47 THE AVIAN CHORIO-ALLANTOIC MEMBRANE: A SUITABLE SHORT-TERM CULTURE SYSTEM FOR BOVINE OVARIAN TISSUE

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab47 ◽

2015 ◽

Vol 27 (1) ◽

pp. 116

Author(s):

K. L. Beck ◽

J. Singh ◽

M. Anzar

Keyword(s):

In Vitro Culture ◽

Blood Vessels ◽

Culture System ◽

Ovarian Tissue ◽

Short Term ◽

In Ovo ◽

Ovarian Cortex ◽

In Vitro Culture System ◽

Short Term Culture

Successful cryopreservation of bovine ovarian tissue holds enormous potential for long-term maintenance of female gametes to preserve genetic diversity by tissue banking. Traditionally, in vitro culture followed by histopathological examination has been used to assess the post-thaw viability of cryopreserved tissues. Recently, in ovo transplantation of mammalian tissues on the chorio-allantoic membrane (CAM) of a growing chicken embryo has emerged as an alternative method for short-term culture. The purpose of this experiment was to compare CAM culture of bovine ovarian tissue over a 5-day period with the in vitro culture system. Fertilized White Leghorn eggs were incubated at 37°C and 62% relative humidity. A window (1 × 2 cm) was cut into the eggshell on Day 3 of incubation. Ovaries were retrieved from a local abattoir and brought to the laboratory within 6 h. Ovarian cortex pieces (1–2 mm3) were randomly assigned to control, CAM-culture, or in vitro-culture groups. Control-group tissues were fixed immediately in 4% paraformaldehyde. The CAM was traumatized on Day 10 of incubation to expose the underlying blood vessels, and tissue pieces were grafted at the site (one graft per egg). For in vitro culture, the ovarian cortex pieces were placed on tissue culture inserts within 6-well plates containing TCM199 with 1% insulin-transferrin-selenium, 100 mIU mL–1 of FSH, 100 IU mL–1 of penicillin, and 50 μg mL–1 of streptomycin and incubated at 38°C in 5% CO2. Ovarian tissues from the CAM and in vitro culture group were removed on Day 1, 3, and 5 of grafting/culture, fixed, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin-eosin, and analysed under a light microscope. The numbers of normal and degenerated follicles (indicating follicle survival) and number of blood vessels containing bovine and avian red blood cells (indicating angiogenesis) were counted using standard stereological procedures. All ovarian cortex grafts from surviving chick embryos showed adhesion with the CAM and a marked neo-vascularization in the graft areas. Gross and histological examination revealed the circulation of avian blood cells in ovarian stromal vessels with a concomitant decrease in the number of bovine blood vessels over the incubation period. Total follicle densities (mean ± s.e.m.) on Day 1, 3, and 5 were 13.3 ± 5.9, 27.9 ± 6.7, and 36.9 ± 7.3 in the in vitro-cultured group and 36.7 ± 13.0, 73.6 ± 24.0, and 44.02 ± 12.67 per millimeter cubed in the CAM-cultured group, respectively. Overall, total follicle density was higher in the CAM-cultured group (P < 0.05). Likewise, the normal follicle densities on Day 1, 3, and 5 were 10.4 ± 4.9, 15.5 ± 3.6, and 20.7 ± 6.3 in the in vitro-cultured group and 30.5 ± 8.5, 45.7 ± 18.4, and 22.7 ± 7.3 per millimeter cubed in the CAM-cultured group (P > 0.05). In conclusion, in ovo CAM grafting system was as successful as the in vitro-culture system and may be considered an acceptable alternative to the traditional in vitro-culture system for bovine ovarian tissue.

Download Full-text

Bioengineering of Human Artificial Ovary From Cryopreserved Tissue: Optimization of the Follicles Enzymatic Isolation.

10.21203/rs.3.rs-190433/v1 ◽

2021 ◽

Author(s):

Jing Chen ◽

Evgenia Isachenko ◽

Wanxue Wang ◽

Xinxin Du ◽

Mengying Wang ◽

...

Keyword(s):

Oxidative Stress ◽

In Vitro Culture ◽

Enzymatic Digestion ◽

Primordial Follicles ◽

Ovarian Cortex ◽

Artificial Ovary ◽

The Difference ◽

Group 2 ◽

Group 1

Abstract Background: This study aims to evaluate the effectiveness of Tumor Dissociation Enzyme (TDE) for the isolation of cryopreserved human ovarian cortex tissues, proposing an optimized applicable for artificial ovary.Methods: This is a prospective experimental study. We present a comparative analysis among the outputs of follicles isolated from cryopreserved ovarian biopsies of ten young women undergoing laparoscopy. Follicles were isolated by Tumor Dissociation Enzyme (TDE) or Liberase Dispase High (DH) enzymatic digestion. Follicles were assessed by evaluating the number, viability, morphology, oxidative stress. Moreover, the follicle growth and viability were analyzed after eight days of in vitro culture (IVC) between each protocol.Results: The recovery rate of follicles in Group 1 was significantly higher by 78 follicles compare to Group 2 (p < 0.05), while the difference in weight and volume of biopsies in both groups was no significant (p > 0.05). Group 1 allowed to isolate more primordial follicles (p < 0.05) and smaller diameter of follicles (p < 0.01) than Group 2. Group 1 allowed to isolate 7% more of bright red follicles than Group 2 (p < 0.01). We also found that Group 1 had isolated a significantly higher percent of viable follicles (p < 0.05), more morphologically normal follicles (p < 0.05), and lower oxidative stress levels compared with Group 2 (p < 0.01). The velocity of follicles growth from Day 0 to Day 8 in Group 1 was significantly higher (p < 0.05) than in Group 2. The viability of follicles on Day 8 of in vitro culture in Group 1 was significantly higher than Group 2 (p < 0.05).Conclusions: TDE treatment can be an alternative for Liberase DH, allows the isolation of highly viable follicles from the cryopreserved human ovarian cortex, with an intact morphology and low oxidative stress, and with high proliferation potential after culture in vitro.

Download Full-text