EMAIL THIS PAGE TO A FRIEND

Theriogenology

Isolation and transplantation of sturgeon early-stage germ cells.


PMID 25559841

Abstract

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 °C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell-specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate-labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P < 0.05). The efficiency of transplantation was 60% (counted 90 dpt).

Related Materials

Product #

Image

Description

Molecular Formula

Add to Cart

09743
3,3′,5,5′-Tetramethylbenzidine, standard for GC
C16H20N2
F0382
Anti-Rabbit IgG (whole molecule)–FITC antibody produced in goat, affinity isolated antibody, buffered aqueous solution
A7511
Bovine Serum Albumin, lyophilized powder, essentially fatty acid free, ≥97% (agarose gel electrophoresis), 1X Crystallized
C0130
Collagenase from Clostridium histolyticum, for general use, Type I, 0.25-1.0 FALGPA units/mg solid, ≥125 CDU/mg solid
F7250
Fluorescein isothiocyanate isomer I, suitable for protein labeling, ≥90% (HPLC), powder
C21H11NO5S
1295607
Glycerin, United States Pharmacopeia (USP) Reference Standard
C3H8O3
PHR1020
Glycerin, Pharmaceutical Secondary Standard; Certified Reference Material
C3H8O3
49779
Glycerol, tested according to Ph Eur, anhydrous
C3H8O3
77067
Glycerol, analytical standard
C3H8O3
H6648 Hanks’ Balanced Salt solution, Modified, with sodium bicarbonate, without phenol red, calcium chloride and magnesium sulfate, liquid, sterile-filtered, suitable for cell culture
L5520 L-15 Medium (Leibovitz), Without L-glutamine, liquid, sterile-filtered, suitable for cell culture
P1644 Percoll®, pH 8.5-9.5 (20 °C)
84100
Sucrose, for microbiology, ACS reagent, ≥99.0%
C12H22O11
S1600000
Sucrose, European Pharmacopoeia (EP) Reference Standard
C12H22O11
1623637
Sucrose, United States Pharmacopeia (USP) Reference Standard
C12H22O11
PHR1001
Sucrose, Pharmaceutical Secondary Standard; Certified Reference Material
C12H22O11
S1174
Sucrose, analytical standard, for enzymatic assay kit SCA20
C12H22O11
T1426
Trypsin from bovine pancreas, TPCK Treated, essentially salt-free, lyophilized powder, ≥10,000 BAEE units/mg protein