In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs.

Although spermatogonial stem cells (SSC) constitute primary candidates for in vitro germ cell (GC) derivation, they are scarce and difficult to maintain in an undifferentiated state. Alternatively, mesenchymal stem cells (MSC) are also candidates for GC derivation due to their simplicity for culture and multipotential for transdifferentiation. The aim of the present study was to compare the GC differentiation potentials of bull peripheral blood-derived MSC (PB-MSC) and SSC using an in vitro 3D co-culture system with Sertoli cells (SC). Samples of PB-MSC or SSC co-cultures with SC were collected on days 0, 7, 14 and 21 and analyzed for pluripotency, GC and mesenchymal marker expression. Co-culture of PB-MSC+SC resulted in down-regulation of NANOG and up-regulation of OCT4 at day 7. In comparison, co-culture of SSC+SC resulted in consistent expression of NANOG, OCT4 and SOX2 at day 14. During co-culture, SSC+SC increased the expression of DAZL, PIWIL2, FRAGILIS and STELLA and activated the expression of STRA8, whereas co-culture of PB-MSC+SC only increased the expression of DAZL and PIWIL2. Thus, co-culture of bull PB-MSC+SC and SSC+SC in 3D SACS results in differential expression of pluripotency and GC markers, where bull SSC display a more robust GC differentiation profile compared to PB-MSC.

Due to their self-renewal and differentiation potentials, mesenchymal stem cells (MSCs) may be induced into germ cells (GC) differentiation under in vitro conditions. In veterinary medicine, this technology could provide an alternative method to artificial insemination, as well as potentially useful for the conservation of endangered species. Previous studies have reported the use of SCs and MSCs co-culture systems, as well as SCs conditioned medium (SCCM) to induce GC differentiation of human and murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The objective of this study was to evaluate the effect of SCCM as an inducer of in vitro GC differentiation of MSCs derived from fetal bovine adipose tissue (AT-MSCs). SCCM was collected from bovine SC cultures generated from adult bull testis. The effect of SCCM on MSCs was analyzed using quantitative PCR (Q-PCR) and flow cytometry. CD73 mRNA levels were decreased (P0.05) between treated and controls cells. DAZL gene expression was detected on day 21 of culture, as well as a proportion of AT-MSC positive for DAZL at day 21 of culture. OCT4, PIWIL2 and DAZL gene expressions were detected from day 0, 7 and 21 of culture, respectively, as well as a proportion of cells positive for each marker were detected at day 21 of culture. However, similar gene and protein expression levels (P>0,05) were detected between AT-MSCs/SCCM and control cultures. DMC1 gene expression levels were detected from day 7 of culture, and expression levels were not different (P>0,05) between treatment and control cells. Expression patterns of MSC, pluripotent, GC and meiotic markers indicate that SCCM did not induce GC differentiation of AT-MSCs.

Organoids serve as three-dimensional structures that offer spatial support and mimic cellular niches for experimental studies. The spatial and temporal association of fetal bovine adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) with the expression of germ cell (GC) markers was evaluated in testicular organoids (TOs+AT-MSCs) under the effect of retinoic acid and testosterone (0.2, 2 and 20 µM). TOs+AT-MSCs were developed by the co-culture of Leydig, Sertoli, and peritubular myoid cells and PKH26+AT-MSCs and were analyzed for the expression of DAZL, STRA8 and UCHL1. TOs+AT-MSCs treated with 1 µM of retinoic acid and 2 or 20 µM of testosterone maintained their diameter after day 7 of culture. Pearson correlation coefficients (PCCs) of PKH26 and DAZL were higher (p < 0.05) at days 7 and 21 in TOs+AT-MSCs treated with 1 µM retinoic acid and 0.2 or 2 µM of testosterone, respectively. PCCs of PKH26 and STRA8 or UCHL1 were higher (p < 0.05) at days 14 and 21 in TOs+AT-MSCs treated with 1 µM retinoic acid and 0.2 or 2 µM of testosterone. Thus, exposure to 1 µM of retinoic acid and 2 µM of testosterone resulted in a similar diameter with respect to the control and spatial and temporal association of AT-MSCs and DAZL, STRA8 and UCHL1 markers in TOs+AT-MSCs.