Hyakutake K et al. (2015), Asymmetrical allocation of JAK1 mRNA during spe...

XB-ART-50713

Dev Growth Differ 2015 Jun 01;575:389-399. doi: 10.1111/dgd.12219.

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Asymmetrical allocation of JAK1 mRNA during spermatogonial stem cell division in Xenopus laevis.

Hyakutake K , Kawasaki T , Zhang J , Kubota H , Abe SI , Takamune K .

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During Xenopus spermatogenesis, each primary spermatogonium (PG), the largest single cell in the testis, undergoes mitotic divisions with a concomitant decrease in size to produce smaller differentiating spermatogonia. The spermatogonial stem cells (SSCs) occur in this PG population. Taking advantage of identifiable and isolatable properties of Xenopus SSCs, we examined JAK1 gene expression during the spermatogenesis because there have been reports on the important role of JAK/STAT pathway in regulating the status of SSCs in Drosophila and mouse. Surprisingly, in situ hybridization revealed the presence of JAK1 mRNA in the differentiating spermatogonia and primary spermatocytes as well as some PGs. Inhibition of JAK1 activity in the testis caused a decrease in percentage of BrdU-incorporating spermatogonia, suggesting that JAK1 was at least involved in regulation of spermatogonial proliferation. Interestingly, single cell reverse transcription-polymerase chain reaction (RT-PCR) clearly showed two different types of SSCs: SSCs with JAK1 mRNA (JAK1+ ) or without JAK1 mRNA (JAK1- ). Since JAK1- SSC level was increased by induction of testis regeneration, self-renewing SSCs were thought to be JAK1- . In addition, we found barrel-shaped PGs, in which JAK1 mRNA was localized asymmetrically to one half of the cell. The stainability with propidium iodide and morphology of two nuclei in the barrel-shaped PG were similar to those of PG nucleus. Based on the above observations, we propose the hypothesis that JAK1+ SSC is preparing for production of PGs destined to differentiate (destined PGs) and the accumulated JAK1 mRNA in the SSC is distributed exclusively into the destined PGs through mitotic division.

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Species referenced: Xenopus laevis
Genes referenced: actl6a fshb gfra1 jak1 jak2 neurog3 tdrd6
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Phenotypes: Xla Wt + AZD1480 (fig.4) [+]

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	Fig. 1. Expression of JAK1 mRNA in testis. Reverse transcription using total testis RNA as a template was carried out with (RT+) or without (RT–) transcriptase. cDNA fragments encoding JAK1, Xtr, and actin were amplified by polymerase chain reaction (PCR), followed by the electrophoresis on 1% agarose gel and staining with ethidium bromide.
	Fig. 2. Images of spermatogenic cells in the section. The sections fixed with Bouin’s fixative (A and A’) and 4% paraformaldehyde in phosphate-buffered saline (PBS) (B and B’) were immunostained with anti-Xtr antiserum (A’ and B’), followed by staining with hematoxylin and eosin (A and B). a, a’, b, and b’ are enlarged images of the areas surrounded by the rectangles in A, A’, B, and B’, respectively. PG, primary spermatogonium; SG, secondary spermatogonia; PC, primary spermatocytes; RT, round spermatids; Scale bars indicate 50 lm in A, A’, B, and B’; and 20 lm in a, a’, b, and b’.
	Fig. 3. Expression of JAK1 mRNA in adult testis. The sections of the testis fixed with 4% paraformaldehyde in phosphatebuffered saline (PBS) were treated with antisense (A) or sense (C) probe for JAK1 mRNA. After detection of the signals, the sections were stained with hematoxylin and eosin (A’, C’). A section adjacent to the antisense probe-treated section was immunostained with anti-Xtr antiserum to detect spermatogenic cell (B). a, a’, and b are enlarged images of the areas surrounded by the rectangles in A, A’ and B, respectively. PG, primary spermatogonium; SG, secondary spermatogonia; PC, primary spermatocytes; RT, round spermatids; MS, mature sperm. Note that PGs are distinguished by the presence (arrowheads in A and a) or absence (double arrowhead in A and a) of JAK1 mRNA. Scale bars indicate 50 lm in A, A’, B, C and C’; and 20 lm in a, a’, and b.
	Fig. 4. Effect of AZD1480 on the efficiency of BrdU-incorporation into spermatogenic cell. After cultivation of the testes fragments successively in follicle-stimulating hormone (FSH)- supplemented medium with (5, 10, and 20 lmol/L) or without (0 lmol/L) AZD1480 for 2 days and in the same medium containing BrdU for 6 h, the number of primary spermatogonia (PG) and cysts consisting of secondary spermatogonia (SG) with or without BrdU in their nuclei was counted and their proliferating activity was assessed by calculating the percentage of BrdUpositive PG and SG. Each data represents a mean of three independent experiments (PG: 0 lmol/L n = 18, 16, 18; 5 lmol/L n = 22, 15, 8; 10 lmol/L n = 15, 13, 14; 20 lmol/L n = 20, 19, 23 and SG: 0 lmol/L n = 6, 7, 6; 5 lmol/L n = 8, 6, 7; 10 lmol/ L n = 8, 7, 10; 20 lmol/L n = 7, 6, 6) with a standard deviation indicated by a bar. The difference between two values was analyzed by Student’s t-test and statistical significance was shown as P-value <0.01 (*) or 0.05 ().
	Fig. 5. Expression of JAK1 mRNA in juvenile testis. A and A’: Section of the testis was treated with antisense probe for JAK1 mRNA (A). After detection of the signals, the section was stained with hematoxylin and eosin (A’). Note that primary spermatogonia (PGs) are distinguished by the presence (arrowheads) or absence (double arrowheads) of JAK1 mRNA. Scale bars indicate 20 lm. B: Each isolated primary spermatogonium (PG) and secondary spermatogonium (SG) from the juvenile testis was examined for JAK1 mRNA by single cell Reverse transcription- polymerase chain reaction and each band was examined for whether specific DNA fragments had been amplified by Southern hybridization. Note that two (lanes 2 and 6) out of nine PGs did not express JAK1 mRNA. Xtr, germ cell marker, and actin were also run as internal controls.
	Fig. 6. Expression of JAK1 mRNA in spermatogonial stem cells. By single cell reverse transcription–polymerase chain reaction (RT–PCR), the presence of JAK1 mRNA was examined in each isolated spermatogonial stem cell (SSC) from the juvenile testes that were cultured in a medium supplemented with follicle-stimulating hormone for 2 weeks. Note that five (lanes 2, 4, 6, 7, and 8) out of 10 SSCs did not express JAK1 mRNA. Xtr, germ cell marker, and actin were also run as internal controls. Each bands was examined for whether specific DNA fragments had been amplified by Southern hybridization.
	Fig. 7. Expression of JAK1 mRNA in regenerating testis. (A) A 5.0 mm long adult testis before surgery. (B) The same testis shortened to 2.2 mm by partial removal. (C) The same testis recovered to 5.5 mm long at 5 weeks after the surgery. Scale bars indicate 2 mm. (D) The ratio of PGs without JAK1 mRNA in the testes before and after induction of regeneration were determined by in situ hybridization using antisense probe for JAK1 mRNA. Each data represents a mean of three independent experiments (before: n = 200, 213, 155 and after: n = 213, 240, 220) with a standard deviation indicated by a bar. The difference between the two values (“before” and “after”) was analyzed by Student’s t-test. The P-value obtained was <0.01 (**) and statistically significant.
	Fig. 8. Asymmetric distribution of JAK1 mRNA in barrel-shaped primary spermatogonia (PGs). Using antisense RNA probe for JAK1 mRNA, JAK1 mRNA in the PGs was detected by in situ hybridization on the sections from Cytochalasin B-treated adult testis (A’, A”, B’, and B”) and Cytochalasin B-treated regenerating adult testis (C’ and D’). The blue signal indicating the presence of JAK1 mRNA was detected only in one side of the barrel-shaped PGs (arrowheads; A’, A”, B’, and B”) or not detected at all (C’ and D’). The double arrowheads indicate a side with no signal. A’ and B’ are higher contrasted images of A” and B”, respectively. After detection of JAK1 mRNA by in situ hybridization, the sections were stained with propidium iodide (A, B, C, and D). D and D’ are enlarged images of the areas surrounded by the rectangles in C and C’, respectively. The barrel-shaped PGs are outlined by the dotted lines (A, B, and D). Note that the barrel-shaped PGs possessed two nuclei having weak stainability with propidium iodide. Scale bars indicate 10 lm in A, A’, A”, B, B’, B”, D, and D’; 20 lm in C and C’.
	Fig. 9. The hypothetical model of the timing of expression and asymmetric distribution of JAK1 mRNA in spermatogenic stem cells (SSCs). The SSC preparing for production of primary spermatogonium (PG) destined to differentiate (Differentiation) but not the self-renewing SSC (Self-renewal) expresses JAK1 mRNA (red dots). The accumulated JAK1 mRNA is distributed exclusively into the PGs destined to differentiate into sperm (Destined PG) during the SSC division.