Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.
BACKGROUND
The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture.


METHODS
Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5).


RESULTS
The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001).


CONCLUSIONS
Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.
Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Banafsheh
B
Heidari
Immunology Research Center (IRC), Tabriz University of Medical Science, Tabriz, Iran.
Minoo
M
Gifani
Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran ; Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
Abolfazl
A
Shirazi
Nanobiotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Amir-Hassan
AH
Zarnani
Immunology Research Center (IRC), Tabriz University of Medical Science, Tabriz, Iran.
Behzad
B
Baradaran
Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Mohammad Mehdi
MM
Naderi
Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Bahareh
B
Behzadi
Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Sara
S
Borjian-Boroujeni
Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Ali
A
Sarvari
Nanobiotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Niknam
N
Lakpour
Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Mohammad Mehdi
MM
Akhondi
C-kit; Differential plating; Goat; PGP 9.5; Percoll gradient; Type A spermatogonia