Establishment of a Coilia nasus Spermatogonial Stem Cell Line Capable of Spermatogenesis In Vitro

Abstract

The process by which spermatogonial stem cells (SSCs) continuously go through mitosis, meiosis, and differentiation to produce gametes that transmit genetic information is known as spermatogenesis. Recapitulation of spermatogenesis in vitro is hindered by the challenge of collecting spermatogonial stem cells under long-term in vitro culture conditions. Coilia nasus is a commercially valuable anadromous migrant fish found in the Yangtze River in China. In the past few decades, exploitation and a deteriorating ecological environment have nearly caused the extinction of C. nasus's natural resources. In the present study, we established a stable spermatogonial stem cell line (CnSSC) from the gonadal tissue of the endangered species C. nasus. The cell line continued to proliferate and maintain stable cell morphology, a normal diploid karyotype, and gene expression patterns after more than one year of cell culture (>80 passages). Additionally, CnSSC cells could successfully differentiate into sperm cells through a coculture system. Therefore, the establishment of endangered species spermatogonial stem cell lines is a model for studying spermatogenesis in vitro and a feasible way to preserve germplasm resources.

Keywords: Coilia nasus; cryopreservation; in vitro spermatogenesis; spermatids; spermatogonial stem cell line.

Figure 1

The flow chart of experiments. A total of five 6-month-old C. nasus measuring 12 cm in length were accessed. Live fish were put on ice and did not respond to mechanical stimulation. All gonadal tissues were separated, then twice washed in PBS with 1% antibiotics. They were minced and dissociated by trypsinization. The cells were transferred to liquid nitrogen for cryopreservation through appropriate culture. The cells were verified through chromosomal analysis, alkaline phosphatase (AP) staining, RT-PCR, and immunofluorescence and characterized through induction in vitro (the figure was created by Biorcndcr.com).

Figure 2

C. nasus spermatogonial stem cells (CnSSCs) were cultured in vitro. (A) The cells were initially dissociated from the gonadal tissue with a large amount of fat (arrow); (B) a small number of C. nasus primary testis cells adhered to the plate on the second day; (C) the cells adhered to the plates, proliferated, and achieved 80% confluency within seven days; (D) primary culture of testis cells on the 11th day. EB: embryoid bodies. (E) Cell morphology on the first day of picking spheroid colonies in culture showing a small size and oval or polygonal shape after dissociation (arrow); (F) subculture of CnSSC cells at passage 78. (Bars = 20 µm unless indicated.)

Figure 3

Alkaline phosphatase staining of CnSSCs. (A–C) Morphology and positive alkaline phosphatase activity staining of cells at P38 (arrow). (D–F) morphology and positive alkaline phosphatase staining of P38 cells after thawing (arrow). The squares of (B(E)) are showed in (C(F)). (Bars = 20 µm.)

Figure 4

Chromosome analysis, RT-PCR analysis, and immunofluorescence analysis of CnSSCs. (A) Diploid metaphase of CnSSCs. (B) Expression of germ cell markers, using RT-PCR, of total RNA from CnSSCs and adult tissues with primers for dazl and vasa. (C) Expression of a stem cell marker, using RT-PCR, with primers for nanog. (D) Expression of somatic cell markers, using RT-PCR, with primers for clu and hsd3β. (E) Expression of actin was determined for calibration. (F–K) Immunofluorescence of PCNA (red) and Vasa (green) in CnSSCs. (Bars = 10 µm.)

Figure 5

The red fluorescent protein (RFP)-expressing CnSSCs were cultured in vitro. (A–D) After stable proliferation, CnSSCs cells labeled with RFP were extracted using methods of single-cell clone culturing, exhibiting positive alkaline phosphatase staining (arrow). (E–G) RFP-expressing CnSSCs formed embryoid bodies by coculture with CnGSCs. (Bars = 20 µm.)

Figure 6

Spermiogenic progression and sperm production from CnSSCs induced in vitro. (A–C) CnSSC cells differentiated into spherical sperm for 5 days (A); the tail pulled longer on the 7th and 12th days (B,C). (A’’–C’’) Merge of bright and fluorescent fields. (Bars = 5 µm.)

Figure 7

RT-PCR analysis of CnSSCs. Expression of meiotic gene markers, using RT-PCR, of total RNA from CnSSCs and adult tissues with primers for dmc1 and rec8 in vitro. Expression of dmc1 and rec8 transcripts is low in undifferentiated CnSSCs. CnSSCs express both genes in the coculture condition. K: kidney; O: ovary; T: testis.