Overexpression of STRA8, BOULE, and DAZL Genes Promotes Goat Bone Marrow-Derived Mesenchymal Stem Cells In Vitro Transdifferentiation Toward Putative Male Germ Cells

Abstract

Bone marrow mesenchymal stem cells (BMSCs), which are well characterized and widely utilized adult stem cells, encompass the capacity to commit to a variety of cell types. This study was conducted to develop an effective way to induce goat BMSCs (gBMSCs) to transdifferentiate toward putative male germ cells by overexpressing STRA8 (stimulated by RA-8), BOULE (also called BOLL), and DAZL (deleted in azoospermia-like). First, we found that the expression levels of these 3 genes gradually increased during development of the goat testis from 10 days postnatal to 8 months old. Therefore, we hypothesized that overexpressing these genes might contribute to the transdifferentiation of gBMSCs toward germ cells. We then overexpressed, separately and in combination, STRA8, BOULE, and DAZL in gBMSCs. Our results showed that a small population of transfected gBMSCs transdifferentiated into early goat germ cell-like cells and that these cells expressed primordial germ cell specification genes STELLA (also known as DPPA3, developmental pluripotency associated 3) and C-KIT (tyrosine kinase receptor) as well as premeiotic genes MVH (mouse vasa homolog), DAZL, BOULE, STRA8, PIWIL2 (piwi-like RNA-mediated gene silencing 2), and RNF17 (ring finger protein 17). Importantly, results from quantitative reverse transcription polymerase chain reaction, immunofluorescence, and Western blot analysis showed that the meiotic marker synaptonemal complex protein 3 (SCP3) significantly increased in transfected cells compared to untransfected control cells ( P < .05). Additionally, the co-overexpression group cells had the highest SCP3 messenger RNA and protein expression levels, which indicated that 3-gene co-overexpression had the highest potential to transdifferentiate gBMSCs to germ cells. Taken together, these results demonstrate that the overexpression of STRA8, BOULE, and DAZL was able to promote the transdifferentiation of gBMSCs to early goat germ cell-like cells in vitro, which probably enhanced maturation and progression through meiosis. This approach would be important to generating gametes for future basic science as well as for potential clinical applications.

Histomorphological Comparisons and Expression Patterns of BOLL Gene in Sheep Testes at Different Development Stages

“…In mice, Zhang et al [9] report that Boll expression is barely detectable from postnatal 3- to 14-day-old testes, but its expression was significantly upregulated at 12-days-old and later. In goats, the expression of BOLL in the testes was observed to be lacking, or low, during the embryonic and pre-pubertal stages, but gradually increased during postnatal progression with a significant expression in mature testes [25,26]. Moreover, BOLL expression in adult dairy goat testes with complete spermatogenesis was significantly higher than that in testes with azoospermia or male intersex [25], which demonstrates that BOLL is crucial for male fertility.…”

“…Li et al [25] reported that the overexpression of BOLL in male dairy goat germline stem cells can elevate the expression of meiosis-associated genes, such as STRA8 , SCP3 , CDC25A , CDC2, and VASA, promoting meiosis and spermatogenesis. In vitro, overexpression of BOLL may effectively promote the transdifferentiation of goat bone marrow mesenchymal stem cells into early germ cells through spermatogenesis [26]. Additionally, experiments in male mice have proven that Boll is involved in regulating the formation of stress granules (SGs) in germ cells under heat stress, to protect those cells from heat-induced apoptosis [27].…”

Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

“…Dazl , an intrinsic meiotic competence factor, is required for Stra8 -mediated initiation of meiosis in germ cells ( Lin et al, 2008 ). Overexpression of Stra8 and Dazl genes promotes the transdifferentiation of mesenchymal stem cells and ESCs in vitro toward PGCs ( Li et al, 2017 ; Shi et al, 2014 ). The elevated expression of Pou5f1 , Dazl , Stella and Stra8 might indicate that the GFP positive cells separated from teratomas partially develop towards germ cells.…”

Functional Germ Cells From Non-Testicular Adult Stem Cells: A Dream or Reality?

“…Moreover, cooverexpression of the three genes had the highest inductive effect for differentiation into GCs. The authors indicated that all stages of GC development process from PGCs to post meiotic stage had occurred in the culture, though they did not further check the cells for DNA content and fertilizing capacity [55].…”