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. 2009 Nov 19:7:130.
doi: 10.1186/1477-7827-7-130.

Gene expression profiles of mouse spermatogenesis during recovery from irradiation

Affiliations

Gene expression profiles of mouse spermatogenesis during recovery from irradiation

Fozia J Shah et al. Reprod Biol Endocrinol. .

Abstract

Background: Irradiation or chemotherapy that suspend normal spermatogenesis is commonly used to treat various cancers. Fortunately, spermatogenesis in many cases can be restored after such treatments but knowledge is limited about the re-initiation process. Earlier studies have described the cellular changes that happen during recovery from irradiation by means of histology. We have earlier generated gene expression profiles during induction of spermatogenesis in mouse postnatal developing testes and found a correlation between profiles and the expressing cell types. The aim of the present work was to utilize the link between expression profile and cell types to follow the cellular changes that occur during post-irradiation recovery of spermatogenesis in order to describe recovery by means of gene expression.

Methods: Adult mouse testes were subjected to irradiation with 1 Gy or a fractionated radiation of two times 1 Gy. Testes were sampled every third or fourth day to follow the recovery of spermatogenesis and gene expression profiles generated by means of differential display RT-PCR. In situ hybridization was in addition performed to verify cell-type specific gene expression patterns.

Results: Irradiation of mice testis created a gap in spermatogenesis, which was initiated by loss of A1 to B-spermatogonia and lasted for approximately 10 days. Irradiation with 2 times 1 Gy showed a more pronounced effect on germ cell elimination than with 1 Gy, but spermatogenesis was in both cases completely reconstituted 42 days after irradiation. Comparison of expression profiles indicated that the cellular reconstitution appeared equivalent to what is observed during induction of normal spermatogenesis.

Conclusion: The data indicates that recovery of spermatogenesis can be monitored by means of gene expression, which could aid in designing radiation treatment regimes for cancer patients leading to better restoration of spermatogenesis.

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Figures

Figure 1
Figure 1
Correlation between testis weight (mg) and time (days) after irradiation. The testes of mice were locally exposed to radiation with 1 Gy (black columns) or 2 × 1 Gy with an interval of 7 days (light grey columns). The testes were weighted and sampled on the indicated days. For 2 × 1 Gy the days correspond to days after the second dose. Error bars represent standard deviation of the mean of the testes (n = 2-8) removed at the indicated day.
Figure 2
Figure 2
Gene-expression data during postnatal development. Fragments from DDRT-PCR reactions are displayed on long polyacrylamide gels and transcripts identified by sequencing. Intensity of bands was quantified by phosphor imaging. A) Autoradiogram displaying a band corresponding to Dazl, a spermatogonia and spermatocyte-specific gene and below quantification of the band. B) Autoradiogram and quantification of bands corresponding to Vps26a, Gata1 and Ribc2 all generated by the same combination of primers and thus displayed on the same gel. Vps26a and Ribc2 are both examples of genes expressed in pachytene spermatocytes, while Gata1 is expressed in Sertoli cells. C) Autoradiogram and quantification of the spermatid-specific gene, Tnp2.
Figure 3
Figure 3
Expression profiles after irradiation with 1 Gy. Autoradiograms and quantifications after irradiation with 1 Gy. The same genes as is figure 2 are displayed: A) Dazl, B) Vps26a, Gata1, Ribc2, and C) Tnp2. Grey areas indicate roughly the absence of the cell types that express the gene, except for Gata1. Below an estimation of the cell types affected by irradiation as spermatogenesis recovers. Open circles indicate that the indicated cell type is present, while filled circles indicate that the indicated cell types are absent. Grey circles indicate that the cell type is partial present.
Figure 4
Figure 4
Expression profiles during recovery after fractionated irradiation. Autoradiograms and quantifications after fractionated irradiation with two times 1 Gy. The same genes as is figure 2 and 3 are displayed: A) Dazl, B) Vps26a, Gata1, Ribc2, C) Tnp2.
Figure 5
Figure 5
in situ hybridization analysis of Tnp2 expression during recovery after irradiation. ISH analysis of Tnp2 expression during recovery in adult mouse testis after irradiation with 1 Gy. A control, pi days 14, 24 and 38 were shown. Low magnification images of whole testis are shown (left) together with a higher magnification of a representative part of the testis (right). The bars correspond to 100 μm. ISH from additional pi days can be found in Additional file 3.
Figure 6
Figure 6
Schematized gene expression profiles from different cell types. The curves describes the profile of a gene expressed in the given cell type as observed after irradiation with 1 Gy. Above descriptions of the main cellular events is outlined. The relative intensity of expression signal cannot be compared between cell types and will in addition depend on how abundant the transcripts in question are expressed, while the relative profile should fit if expression is confined to the cell type described. The signal from spermatocytes originates primarily from large pachytene spermatocytes, which has a tremendous effect on the expression signal observed from other cell types.

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