Short-term culture of ovarian cortical strips from capuchin monkeys (Sapajus apella): a morphological, viability, and molecular study of preantral follicular development in vitro

Abstract

The aim of this study was to evaluate whether an in vitro culture (IVC) medium containing either or not β-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) could be able to promote the development of capuchin monkeys' preantral follicles enclosed in ovarian cortical strips. Follicular viability after IVC was similar to control (89.32%). Primordial follicle recruitment to primary stage was not reached with IVC, but the rate of secondary follicle formation was increased in the medium supplemented with BME, BMP4, and PMSG (44.86%) when compared to IVC control (9.20%). In the medium supplemented with BME, BMP4, and PMSG, contrary to other media, anti-müllerian hormone-messenger RNA (mRNA) expression in ovarian tissue was upregulated (3.4-fold), while that of growth differentiation factor-9 was maintained. The BMP4-mRNA expression, however, appeared downregulated in all cultured tissues. Our findings show a favorable effect of BME, BMP4, and PMSG on the in vitro development of secondary follicles from capuchin monkeys.

Keywords: growth factors; nonhuman primates; qRT-PCR; viability.

Figure 1.

Experimental protocol for the IVC of capuchin monkeys’ preantral follicles enclosed in ovarian tissue fragments. Viability analysis was performed by labeling preantral follicles with a mixture of propidium iodide (dead cell fluorescent marker) and Hoescht (total cells fluorescent marker). The qRT-PCR was performed to quantify the relative expression (fold-change) of the genes encoding AMH, CTGF, BMP4, BMP15, KL, and GDF9. AMH indicates anti-müllerian hormone; BMP4, bone morphogenetic protein 4; BMP15, bone morphogenetic protein 15; CTGF, connective tissue growth factor; GDF9, growth differentiation factor-9; IVC, in vitro culture; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.

Figure 2.

Panel A-C, Representative images of 2 preantral follicles from capuchin monkeys before (A) and after (B, C) staining with Hoechst (blue; general nuclear staining) and propidium iodide (red; stains dead cells). Absence of propidium iodide staining in the left follicle (C) indicates its viability, whereas the presence of propidium iodide in the right follicle (C) is indicative of its death. ×400. For reference to color, please see online version.

Figure 3.

Rates of viable primordial, primary, and secondary follicles before and after in vitro culture. Follicles were considered viable when a viable oocyte was surrounded by ≥90% viable granulosa cells. Follicles were classified as primordial when the oocyte was surrounded by a layer of flattened granulosa cells, primary when the oocyte was surrounded by 1 layer of cuboidal granulosa cells, or as secondary when the oocyte was surrounded by 2 or more layers of cuboidal granulosa cells. *Indicates significant differences between control group and treatments in each follicular class (P < .05).

Figure 4.

Comparison of mean (± SEM) relative expression of AMH, CTGF, BMP4, BMP15, KL, and GDF9 in ovarian tissue from the control, IVC control group, and ovarian tissue that was cultured in vitro in the presence of BME, BMP4, or PMSG alone or in mixtures of these compounds. Fold-changes in relative mRNA expression were quantified relative to the expression in the control group by qRT-PCR analyses (P <.01). Data were normalized using 2 housekeeping genes (HPRT1 and TBP). AMH indicates anti-müllerian hormone; BME, β-mercaptoethanol; BMP4, bone morphogenetic protein 4; BMP15, bone morphogenetic protein 15; CTGF, connective tissue growth factor; GDF9, growth differentiation factor 9; HPRT1, hypoxanthine phosphoribosyltransferase 1; KL, kit ligand; IVC, in vitro culture; PMSG, pregnant mare serum gonadotrophin; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SEM, standard error of the mean; TBP, TATA box binding protein.