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Synergistic Promoting Effects of X-Linked Inhibitor of Apoptosis Protein and Matrix on the In Vitro Follicular Maturation of Marmoset Follicles

Abstract

BACKGROUND:

In vitro follicular maturation (IVFM) of ovarian follicles is an emerging option for fertility preservation. Many paracrine factors and two-dimensional or three-dimensional (3D) environments have been used for optimization. However, since most studies were conducted using the murine model, the physiological differences between mice and humans limit the interpretation and adaptation of the results. Marmoset monkey is a non-human primate (NHPs) with more similar reproductive physiology to humans. In this study, we attempted to establish a 3D matrix (Matrtigel)-based IVFM condition for marmoset ovarian follicles in combination with anti-apoptotic factor, X-linked inhibitor of apoptosis protein (XIAP).

METHODS:

Marmoset follicles were isolated as individual follicles and cultured in a single drop with the addition of 0, 10, and 100 μg/mL of XIAP molecules. Matured oocytes and granulosa cells from mature follicles were collected and analyzed. The average number of isolated follicles was less than 100, and primordial and antral follicles were abundant in the ovaries.

Results

IVFM of marmoset follicles in 3D matrix conditions with XIAP increased the rates of survival and in vitro follicle development. Furthermore, oocytes from the 3D cultures were successfully fertilized and developed in vitro. The addition of XIAP increased the secretion of estradiol and aromatase. Furthermore, expression of granulosa-specific genes, such as bone morphogenetic protein 15, Oct4, and follicle-stimulating hormone receptor were upregulated in the in vitro-matured follicles than in normal, well-grown, and atretic follicles. Apoptosis-related B-cell lymphoma-2 was highly expressed in the atretic follicles than in the XIAP-treated follicles, and higher caspase-3 was localized in the XIAP-treated follicles.

Conclusion

In this study, we attempted to establish a 3D-matrix-based marmoset IVFM condition and demonstrated the synergistic effects of XIAP. The use of a 3D matrix may be applied as an optimal culture condition for marmoset ovarian follicles.

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Fig. 1

The ovaries or female reproductive organs (FROs) are excised under anesthesia. Isolated ovaries are cut into pieces, and the follicles are isolated. All images are captured under a stereo microscope except for A. A excised FROs of marmoset monkeys; B isolated ovaries; C sliced half ovaries; D pieces of the ovaries and follicles within the ovarian tissue are imaged inside the circles

Fig. 2

Histological observations of sectioned marmoset ovaries. The images of the follicles of each stage are enlarged in a blue-lined circle

Fig. 3

Isolated marmoset ovarian follicles are subjected to in vitro-maturation in a 2D or 3D environment with or without Xlinked inhibitor of apoptosis protein (XIAP). The in vitro development of the follicles is evaluated by changes in morphology with filled follicular fluid. Representative images of follicles are taken on days 3, -8, and -13. A in vitro follicular growth in 2D; B in vitro follicular growth in 3D

Fig. 4

In vitro ovulation is induced by treatment with human chorionic gonadotropin (hCG), and movement to the follicle membrane region is observed. A ruptured oocyte is collected, and mature oocytes (MII) are further processed for in vitro fertilization (IVF). Fertilized eggs develop into the cleavage stage and into eight-cell stage in vitro. Representative images of A are taken on day 14. A Induction of ovulation by hCG treatment; B IVF of oocytes from in vitro follicular maturation

Fig. 5

Secretion of estradiol (E2) and aromatase were evaluated by enzyme-linked immunosorbent assay. The specific expression of these genes was analyzed by quantitative reverse transcription-polymerase chain reaction. A concentration of E2 in in vitro matured marmoset ovarian follicles; B concentration of aromatase; C specific relative expression of granulosa cell-specific genes (p < 0.0001); D localization of follicle-stimulating hormone receptor (FSH Rc) in follicles grown in vitro

Fig. 6

The expression of anti-apoptotic molecules is analyzed at mRNA and protein levels. A localization of caspase-3; B expression of Bcl-2

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Funding

This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (2019R1A2C1010163 and 2020R1A2C1010293).

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Correspondence to Seung-Yup Ku.

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No potential conflict of interest was reported by the author(s).

Ethical statement

All animal experiments were approved by the IACUC of Seoul National University Hospital [SNUH IACUC No. 15–0032-C2A0(1)].

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Kim, Y.Y., Yun, JW., Kim, S.W. et al. Synergistic Promoting Effects of X-Linked Inhibitor of Apoptosis Protein and Matrix on the In Vitro Follicular Maturation of Marmoset Follicles. Tissue Eng Regen Med (2021). https://doi.org/10.1007/s13770-021-00387-4

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Keywords

  • In vitro follicular maturation
  • Ovarian follicle
  • 3D
  • Marmoset
  • XIAP