Bioengineering Approaches to Improve In Vitro Performance of Prepubertal Lamb Oocytes.

Antonella Mastrorocco,Giacomina Brunetti,Letizia Temerario,Ludovica Cacopardo,Domenico Robbe,Augusto Carluccio,Daniela Lamanna,Maria Elena Dell’Aquila

doi:10.3390/cells10061458

Antonella Mastrorocco, Giacomina Brunetti + Show 6 more

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https://doi.org/10.3390/cells10061458

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Abstract

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET.

Highlights

Oocytes from prepubertal lambs represent a unique model to study oocyte modifications occurring in the period between birth and puberty
The greatest limitation of prepubertal lamb oocytes’ use in Juvenile in vitro embryo technology (JIVET) is that their developmental competence is lower in comparison to oocytes derived from their adult counterpart [13]—a phenomenon observed in other species [13,14,15,16,17]
Bioengineering strategies are becoming widespread in the area of cell culture, including assisted reproductive technologies (ARTs), to the best of our knowledge such approaches have never been applied in prepubertal oocytes in studies conducted to date, but only in adult oocytes [36,51,58,59,60,61,62]

Summary

Introduction

Oocytes from prepubertal lambs represent a unique model to study oocyte modifications occurring in the period between birth and puberty These oocytes are used for JIVET (juvenile in vitro embryo technologies), a promising set of assisted reproductive technologies (ARTs) providing exciting opportunities in different application fields of animal reproduction. The greatest limitation of prepubertal lamb oocytes’ use in JIVET is that their developmental competence is lower in comparison to oocytes derived from their adult counterpart [13]—a phenomenon observed in other species [13,14,15,16,17] This lower embryo developmental competence of oocytes from juvenile ewes has to date been mostly related to incomplete or perturbed in vitro cytoplasmic, molecular, and nuclear maturation. It has been shown that oocytes from prepubertal animals exhibit ultrastructural, molecular, and functional differences compared to adult ones [18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]

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