Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice

Abstract

Aggressive chemotherapy may lead to permanent male infertility. Prepubertal males do not generate sperm, but their testes do contain spermatogonial cells (SPGCs) that could be used for fertility preservation. In the present study, we examined the effect of busulfan (BU) on the SPGCs of immature mice, and the possible induction of the survivor SPGCs to develop spermatogenesis in 3D in-vitro culture. Immature mice were injected with BU, and after 0.5–12 weeks, their testes were weighed and evaluated histologically compared to the control mice. The spermatogonial cells [Sal-like protein 4 (SALL4) and VASA (a member of the DEAD box protein family) in the testicular tissue were counted/seminiferous tubule (ST). The cells from the STs were enzymatically isolated and cultured in vitro. Our results showed a significant decrease in the testicular weight of the BU-treated mice compared to the control. This was in parallel to a significant increase in the number of severely damaged STs, and a decrease in the number of SALL4 and VASA/STs compared to the control. The cultures of the isolated cells from the STs of the BU-treated mice showed a development of colonies and meiotic and post-meiotic cells after four weeks of culture. The addition of homogenates from adult GFP mice to those cultures induced the development of sperm-like cells after four weeks of culture. This is the first study demonstrating the presence of biologically active spermatogonial cells in the testicular tissue of BU-treated immature mice, and their capacity to develop sperm-like cells in vitro.

Effect of Gosha-Jinki-Gan on Levels of Specific mRNA Transcripts in Mouse Testes after Busulfan Treatment

“…Furthermore, we compared the expression levels of mRNA species encoding premeiotic cells ( cKit, Gfra1, and Vasa ), meiotic and postmeiotic cells ( Boll, Crem, Prm1, and Acrosin ) ( Figure 1 C), the spermatogonial marker Stra8 , the spermatocyte marker of Spo11, and the spermatid marker Tnp1 ( Figure 1 B) [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ] by real-time RT-PCR analysis in each group at day 120. The levels of all examined ten spermatogenesis markers were significantly reduced in group III but completely recovered in group V; the expression recovered slightly in group IV but remained significantly less than that in group V and normal mice.…”

“…The mixtures were stored at −80 °C until analysis. Real-time RT-PCR was performed on 2.5-μg cDNA using the validated SYBR Green gene expression assay in combination with SYBR Premix Ex Taq TM (TaKaRa, Bio Inc., Ohtsu, Japan) for the 10 germ cell genes ( Stra8, Spo11, Tnp1, cKit, Gfra1, Vasa, Boll, Crem, Prm1, and Acrosin ), 25 Sertoli cell genes ( Amh, Aqp8, Ccnd2, Clu, Cldn11, Cst12, Cst9, Dhh, Espn, Fshr, Fyn, GATA1, Il1a, Inhba, Inhbb, Msi1, Rhox5, Testin, Shbg, Spata2, Sox9, Tjp1, Trf, Wt1, and Wnt5a ), and GAPDH as an internal control, respectively, with forward and reverse primers described previously [ 15 , 16 , 17 , 18 , 19 ] and was synthesized from Hokkaido System Science Co., Ltd. (Sapporo City, Hokkaido, Japan). Quantitative real-time PCR was performed in duplicate with the Thermal Cycler Dice Real-time System TP800 (TaKaRa), and the thermal profile used for amplification was 95 °C for 30 s followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. The Thermal Cycler Dice Real-time System software (TaKaRa) was used to analyze the data, and the comparative C t method (2∆∆ C t ) was used to quantify the gene expression levels.…”

Exposure to Chemotherapy During Childhood or Adulthood and Consequences on Spermatogenesis and Male Fertility

“…The three-dimensional cell culture and the organotypic culture have been shown to support the differentiation of spermatogonia into spermatozoa in the rat and mouse model [156][157][158][159]. Moreover, the generation of sperm-like cells has been reported in 3D cell culture from spermatogonial cells of busulfan-treated mice [160]. Today, only the organotypic culture allows to produce mature spermatozoa and generate a healthy offspring by intracytoplasmic sperm injection (ICSI) in mice [158,161].…”

Approaches and Technologies in Male Fertility Preservation

“…Our group was the first to suggest the use of a methylcellulose culture system (MCS) and soft-agar-culture system (SACS) as possible three-dimensional (3D) matrices to grow and develop spermatogonial cells in vitro [87][88][89][90][91][92]. Using these two novel 3D culture systems (MCS and SACS), which are more representative of in-vivo conditions, we could induce the proliferation and differentiation of spermatogonial cells from normal and busulfan-treated immature mice to the meiotic and post-meiotic stages, and even the generation of sperm-like cells (elongated spermatid with head, neck, and tail) [87,90,92,93]. The development of meiotic and post-meiotic stages was examined after 3-6 weeks of culture.…”

“…Although in-vitro spermatogenesis has mainly been achieved (not efficiently) in rodent organ culture and/or 3D in-vitro culture systems [27][28][29][30][31][90][91][92][93], the optimal conditions that provide an optimal 3D spatial environment with similar cellular composition of the seminiferous tubule to efficiently induce the development of spermatogonial cells in order to complete maturation and generation sperm was not yet published. A new culture system that provides alginate-based hydrogel and 3D bioprinting was developed to control scaffold design and cell deposition in order to preserve testicular cells in their native 3D spatial and cellular microenvironment to induce complete in-vitro spermatogenesis.…”