64
3.3.1 Collection of Oocyte and Follicular Cells
of the Female Gonads
The collection of the oocyte is easy in animals with extracorporeal fecundation,
such as fish, amphibians, and birds, or non-vertebrates. In mammals, the eggs must
be instead aspirated from the fallopian tubes, a poorly invasive method usually
applied to woman, or picked-up with a needle from a pre-ovulatory follicle.
The granulosa cells and the cumulus–oocyte complex are only available with
puncture and aspiration of mature follicle. (Arashiro et al. 2013). The methods in
use differ widely: those preferred in humans include an ultrasound-guided puncture,
or a laparotomic procedure, generally necessary to obtain gametes for the artificial
fecundation. In livestock and laboratory species, the ovariectomy is more common.
The ovaries are freshly collected after death and safely transported to the laboratory.
To warrant enough yield of reproductive tissue and cells, the ovulation is stimulated
with injection of gonadic tropins shortly before the sacrifice. For experimental
purposes, the cumulus–oocyte complex is aspirated from the antral follicles, better
if the granulosa is three layered. After the isolation, the granulosa cells require
purification on a gradient to avoid the contamination with blood cells (Chilvers
et al. 2012; Ferrero et al. 2012; Quinn et al. 2006). Once isolated and purified,
different types of cells can also be cultivated, stabilized, or immortalized, to obtain
cell lines comparable to those previously cited and commercially available.
In brief, several alternative methods are available to exhaustively study the ovary
function in vitro, all well standardized. They have, however, more limits in
comparison with methods in use to study reproductive toxicity in the male. The low
yield of isolation procedures requires previous hormonal stimulation in vivo, while
the necessary higher manipulation of the functional unit, that is the follicle and its
component, imposes the presence of skilled personnel at the laboratory. Ethical
limits reduce the availability of experimental material from humans.
3.3.2 In Vitro Exposure of Different Types of Ovary Cells
The first study on the safety of nanomaterials for ovary cells was conducted by
Bourrinet et al. 2006, in a complete preclinical study that aimed to ascertain the
eligibility of super paramagnetic iron oxide nanoparticles for clinical aims. The
nanoparticles were safe for the endpoint considered, the induction of chromosomal
aberration in the chinese hamster ovary cells. Further studies confirmed the low
toxicity of these materials in Chinese hamster ovary cells and granulosa cells,
related to the intake inside the cells. Protective coating, such as with dextran, bovine
serum albumin or polyethylene glycol, improved the biocompatibility and safety of
nanoparticles (Hanot et al. 2015; Pöttler et al. 2015, 2016).
Other oxides inhibited the growth of Chinese hamster ovary cells acting on selective genetic pathways (Liu et al. 2017). The effect of cerium oxide nanoparticles
A. G. Cattaneo
3.3.1 Collection of Oocyte and Follicular Cells
of the Female Gonads
The collection of the oocyte is easy in animals with extracorporeal fecundation,
such as fish, amphibians, and birds, or non-vertebrates. In mammals, the eggs must
be instead aspirated from the fallopian tubes, a poorly invasive method usually
applied to woman, or picked-up with a needle from a pre-ovulatory follicle.
The granulosa cells and the cumulus–oocyte complex are only available with
puncture and aspiration of mature follicle. (Arashiro et al. 2013). The methods in
use differ widely: those preferred in humans include an ultrasound-guided puncture,
or a laparotomic procedure, generally necessary to obtain gametes for the artificial
fecundation. In livestock and laboratory species, the ovariectomy is more common.
The ovaries are freshly collected after death and safely transported to the laboratory.
To warrant enough yield of reproductive tissue and cells, the ovulation is stimulated
with injection of gonadic tropins shortly before the sacrifice. For experimental
purposes, the cumulus–oocyte complex is aspirated from the antral follicles, better
if the granulosa is three layered. After the isolation, the granulosa cells require
purification on a gradient to avoid the contamination with blood cells (Chilvers
et al. 2012; Ferrero et al. 2012; Quinn et al. 2006). Once isolated and purified,
different types of cells can also be cultivated, stabilized, or immortalized, to obtain
cell lines comparable to those previously cited and commercially available.
In brief, several alternative methods are available to exhaustively study the ovary
function in vitro, all well standardized. They have, however, more limits in
comparison with methods in use to study reproductive toxicity in the male. The low
yield of isolation procedures requires previous hormonal stimulation in vivo, while
the necessary higher manipulation of the functional unit, that is the follicle and its
component, imposes the presence of skilled personnel at the laboratory. Ethical
limits reduce the availability of experimental material from humans.
3.3.2 In Vitro Exposure of Different Types of Ovary Cells
The first study on the safety of nanomaterials for ovary cells was conducted by
Bourrinet et al. 2006, in a complete preclinical study that aimed to ascertain the
eligibility of super paramagnetic iron oxide nanoparticles for clinical aims. The
nanoparticles were safe for the endpoint considered, the induction of chromosomal
aberration in the chinese hamster ovary cells. Further studies confirmed the low
toxicity of these materials in Chinese hamster ovary cells and granulosa cells,
related to the intake inside the cells. Protective coating, such as with dextran, bovine
serum albumin or polyethylene glycol, improved the biocompatibility and safety of
nanoparticles (Hanot et al. 2015; Pöttler et al. 2015, 2016).
Other oxides inhibited the growth of Chinese hamster ovary cells acting on selective genetic pathways (Liu et al. 2017). The effect of cerium oxide nanoparticles
A. G. Cattaneo