Parâmetros seminais e extração de RNA espermático de mico-leão-de-cara-dourada (Leontopithecus chrysomelas)
Abstract
The Golden-headed lion tamarin (Leontopithecus chrysomelas) is an endemic primate from
Atlantic Forest threatened with extinction. Amongst the conservational efforts addressed
towards the species is the use of reproductive biotechnologies as a strategy of genetic
resources preservation and assurance of captive and natural population´s viability. The
success of the reproductive biotechnologies still depends on the detailed knowledge despite
the reproductive aspects, the investigation of factors that may influence the reproductive
success and the establishment of specific-species protocols. The cryopreservation of semen is
the most widely used approach for wild species. However, one major challenge associated to
the appliance of neotropical primate´s samples consists in the formation of seminal clots,
which invalidates the use of great quantity of gamete in most reproductive procedures. The
golden-headed lion tamarin has become a promising model among threatened neotropical
primates and aiming to contribute with the development of reproductive technologies, this
essay had the objective of initiate investigations concerning the molecular and seminal factor
which may have an effect on the reproductive potential of sexually mature males of
Leontopithecus chrysomelas. Therefore, the animals kept at the Fundação Parque Zoológico
de São Paulo, São Paulo, Brazil, were divided into two groups, “Old” (mean age 11,6 years;
n=6) and “Young” (mean age 4,8 years, n=6) to execute the semen collection and evaluation
of the seminal parameters (concentration, motility, plasmatic and acrosome membrane
integrity, mitochondrial cytochemical activity and morphology). The analysis of seminal
parameters pointed out significant differences between the older and younger subjects despite
the plasmatic membrane integrity (p=0,049), acrosome integrity (p=0,009) and for Class IV
mitochondrial activity (p=0,026). The regression analysis showed that the age, as well as the
weight of the individuals, have a negative influence over the quality of these parameters.
Therefore, aiming to contribute with the development of new reproductive techniques and to
improve the spermatic viability, a protocol of sperm selection by Percoll discontinuous
gradient was tested. However, even though efficient for gamete isolation from other seminal
component, the protocol did not show significant improvement of the parameters, reevaluated
after selection, suggesting needing of methodological adequation for and effective application
in Leontopithecus chrysomelas. To start investigating the gamete´s epigenetic role in male
reproductive success, this essay established a sperm RNA isolation protocol, highlighting the
use of seminal clot, a rich source of genetic material, which is usually thrown away after
processing. The samples of each individual were condensed in two pools, one referring the
clot and other to the liquid fraction. Adaptations for the somatic cellular lyse allowed
obtainment of similar profiles between the two portions of the ejaculate. The qualitative
profiles for sperm RNA of Leontopithecus chrysomelas indicates low concentration of
molecules bigger than RNA, being represented, basically, by little and degraded transcripts
due to silencing of gene expression in spermatozoa. The spermatic transcripts have been
assigned as the determinant function in reproduction, enrolled with the regulation of female
gamete activation and gene expression at the beginning of the embryo development.
Furthermore, is has been proposed that the quantity of RNA present in gametes be used as a
non-invasive tool for deducing male fertility, since these transcripts reflect on events that
occur during cellular differentiation.