Abstract
The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia regardless of the initial stage of tubule development in the donor tissue. Moreover, the number of spermatogonia decreased with time in culture in all media tested, especially in the medium supplemented with KSR, while AlbuMax had a slight protective effect. The combination of AlbuMax and beta-estradiol led to an increase in the area occupied by seminiferous tubules, and thus to an increase in total number of spermatogonial cells. Considering all the media combinations tested the stimulus for felid germ cell differentiation in this type of system seems to be different from the mouse. Studies using other triggers of differentiation and tissue survival factors should be performed to pursue this technology for the genetic diversity preservation in wild felids.
Highlights
Most wild felids are listed as threatened, vulnerable or endangered
The organ culture method allows for the maintenance of the testicular architecture and microenvironment and this is important since the contact between germ cells and somatic cells, Sertoli cells in particular, seems to be critical for the success of the spermatogenic process [6] as they need to be present in order to obtain spermatid-like cells in all the in vitro culture systems developed so far [7,8,9]
Estradiol is a testosterone metabolite produced in Leydig cells and its absence results in progressive loss of fertility and disruption of spermatogenesis [14]
Summary
Most wild felids are listed as threatened, vulnerable or endangered. The loss of genetic diversity is a significant problem in felids given that juvenile mortality of valuable individuals is usually high. Spermatogenesis is a complex process that takes place in the seminiferous epithelium, physical and physiologically regulated by Sertoli, Leydig, and peritubular cells It involves a strict spatial organization, and intricate metabolic reactions. Sato and colleagues [3, 4] reported successful in vitro sperm production after organ culture of immature and adult mouse testis. FSH will directly stimulate Sertoli cells to increase germ cell number and, in turn, Sertoli cells will stimulate Leydig cells This effect, together with the action of LH on Leydig cells, will lead to the production of testosterone required for normal spermatogenesis [11]. It reported that FSH and testosterone had no effect in their organ culture experiments [3] Hormones such as estradiol and progesterone influence male gametogenesis. In Leydig cells, cholesterol leads to the production of progesterone, a metabolic precursor of both androgens and estrogens [15]
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