Applicability of Hyaluronic Acid-Alginate Hydrogel and Ovarian Cells for In Vitro Development of Mouse Preantral Follicles

Abstract

Objective: In the present study, the applicability of hyaluronic acid-alginate (HAA) hydrogel and ovarian cells (OCs) for the culture of mouse ovarian follicles were investigated and compared with those of alginate (ALG) and fibrin-alginate (FA) hydrogels.

Materials and methods: In the first step of this experimental study, mechanically isolated preantral follicles from the ovaries of two-week-old mice were encapsulated in the absence or presence of OCs in ALG, HAA, and FA hydrogels and cultured for 14 days. The morphology, diameter, survival and antrum formation rates of the follicles and the maturation and quality of the oocytes were evaluated during culture. In the second step, preantral follicles were cultured similar to the first step, but for 13 days, and their gene expressions and hormonal secretion were assessed on the last day of culture.

Results: In the absence of OCs, higher numbers of ALG- and HAA-encapsulated follicles reached the antral stage compared to FA-encapsulated follicles (P<0.05). However, a higher percentage of HAA-developed oocytes resumed meiosis up to the germinal vesicle breakdown (GVBD)/metaphase II (MII) stages in comparison with ALG-developed oocytes (P<0.05). HAA-encapsulated follicles had significant overexpression of most of the growth and differentiation genes, and secreted higher levels of estradiol (E2) compared to ALG- and FA-encapsulated follicles (P<0.05). The co-culture condition increased the diameter of ALG-encapsulated follicles on day 13 of culture (P<0.05). It also increased the survival and maturation rates of ALG- and FA-encapsulated follicles, respectively (P<0.05). The co-culture condition improved cortical granule distribution in all groups, increased E2 and progesterone (P4) secretions in the ALG and FA groups, and androstenedione (A4) secretion in the FA group (P<0.05).

Conclusion: The present study results show that HAA hydrogel is a promising hydrogel for follicle culture. OCs utilization could ameliorate the culture conditions regardless of the type of hydrogel.

Keywords: Alginate; Fibrin; Hyaluronic Acid; Ovarian Cells; Preantral Follicle.

Fig.1

Growth of preantral follicles encapsulated and cultured in alginate (ALG), hyaluronic acid-alginate (HAA) and fibrin-alginate (FA) hydrogels in the absence or presence of ovarian cells (OCs and +OCs-respectively). A. Morphological changes and B. Diameters of the surviving follicles on days 1, 6, and 13 of culture. Data are presented as mean diameter ± standard error (SE). Data points a and b differ significantly (P<0.05, scale bar: 100 μm). OCs; ovarian Cells.

Fig.2

Immunofluorescence staining of cortical granules (CGs, red), meiotic spindle (Spdl, green) and chromosomes (Chrs, blue) in normal and abnormal metaphase II (MII) oocytes. The absence of a cortical granulefree domain (CGFD) around the spindle and lack of a cortical distribution, and disorganized spindle configuration or misaligned chromosomes were respectively considered the signs of cortical granules and spindle abnormalities (scale bar: 50 μm).

Fig.3

Expression of growth and differentiation (Gdf9, Bmp15, Zp3, Gja4, Gja1, Bmp4, and Bmp7), apoptotic (Trp53, Casp3, Bax, and Bcl2) and steroidogenic (Fshr, Lhcgr, Cyp11a1, Cyp17a1, and Cyp19a1) genes in follicles encapsulated and cultured in alginate (ALG), hyaluronic acid-alginate (HAA) and fibrinalginate (FA) hydrogels in the absence or presence of ovarian cells (OCs and +OCs-respectively). Antral follicles were collected on day 13 of culture. Expression levels were normalized to GAPDH as the endogenous control. Data are presented as mean ± standard error (SE). Data points A and B, C and D, a and b differ significantly (P<0.05).

Fig.4

Secretion of estradiol (E2), progesterone (P4), and androstenedione (A4) by follicles encapsulated and cultured in alginate (ALG), hyaluronic acid-alginate (HAA) and fibrin-alginate (FA) hydrogels in the absence or presence of ovarian cells (OCs and +OCs-respectively). Conditioned media were collected on day 13 of culture. Data are presented as mean ± standard error (SE). Data points A and B, C and D, a and b differ significantly (P<0.05).