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A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Gupta V, Holets-Bondar L, Roby KF, Enders G, Tash JS - Biomed Res Int (2015)

Bottom Line: Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging.Postfixation processing was also standardized for safe shipment back to our laboratory.Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Mail Stop 3050, 3901 Rainbow Boulevard, HLSIC 3098, Kansas City, KS 66160, USA.

ABSTRACT
Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

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Morphological analysis of mouse epididymis after different postfixative modifications. Sections of adult mouse epididymis were stained with HE (all at 40x; magnification bar is 50 μm). (a) Control; (b) slow rehydration-dehydration stepwise replacement of ETOH-PBS-ETOH; (c) slow Re-ETOH only replacement; (d) rapid ETOH-PBS-ETOH transition. Epithelium (E), sperm (S), basal cell (B), and principal cell (P).
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Related In: Results  -  Collection


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fig5: Morphological analysis of mouse epididymis after different postfixative modifications. Sections of adult mouse epididymis were stained with HE (all at 40x; magnification bar is 50 μm). (a) Control; (b) slow rehydration-dehydration stepwise replacement of ETOH-PBS-ETOH; (c) slow Re-ETOH only replacement; (d) rapid ETOH-PBS-ETOH transition. Epithelium (E), sperm (S), basal cell (B), and principal cell (P).

Mentions: Testicular and epididymis morphology was evaluated for histological changes after different postfixation processing (Figures 4 and 5). In control testicular tissue (Figure 4(a)), all types of spermatogenic cells, spermatogonia (Sp), Sertoli cells (Se), spermatocytes (Sc), and spermatids (Sd), were evident. Lymphatic spaces between seminiferous tubules and adjacent to Leydig cells (L) clusters are clearly defined. After the slow rehydration-dehydration stepwise replacement with ETOH, testis tubules appeared normal with histological architecture similar to the control group (Figure 4(b)). Although all types of spermatogenic cell were identified after slow rehydration-dehydration stepwise replacement, spermatogonia and Sertoli cell nuclei were more difficult to distinguish compared to control. The quality of testicular histology observed after slow Re-ETOH only (Figure 4(c)) was similar to that observed in the slow rehydration-dehydration stepwise replacement group. However, open spaces (black arrows) were observed within portions of seminiferous tubules in the slow Re-ETOH only group, and the seminiferous tubule basement membrane in this group often appeared wavy and thinner (yellow arrows) compared to the control (Figure 4(a)). The histologic quality was poor in the group treated by single step (rapid transition) change of solution (Figure 4(d)), as evidenced by a diffuse appearance of the tissue (not due to focus) and limited clarity of nuclear details in spermatogonia and Sertoli cells. In addition, the seminiferous tubule basement membrane was occasionally indistinct and some spermatid artifactual loss of residual bodies is also observed.


A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Gupta V, Holets-Bondar L, Roby KF, Enders G, Tash JS - Biomed Res Int (2015)

Morphological analysis of mouse epididymis after different postfixative modifications. Sections of adult mouse epididymis were stained with HE (all at 40x; magnification bar is 50 μm). (a) Control; (b) slow rehydration-dehydration stepwise replacement of ETOH-PBS-ETOH; (c) slow Re-ETOH only replacement; (d) rapid ETOH-PBS-ETOH transition. Epithelium (E), sperm (S), basal cell (B), and principal cell (P).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309301&req=5

fig5: Morphological analysis of mouse epididymis after different postfixative modifications. Sections of adult mouse epididymis were stained with HE (all at 40x; magnification bar is 50 μm). (a) Control; (b) slow rehydration-dehydration stepwise replacement of ETOH-PBS-ETOH; (c) slow Re-ETOH only replacement; (d) rapid ETOH-PBS-ETOH transition. Epithelium (E), sperm (S), basal cell (B), and principal cell (P).
Mentions: Testicular and epididymis morphology was evaluated for histological changes after different postfixation processing (Figures 4 and 5). In control testicular tissue (Figure 4(a)), all types of spermatogenic cells, spermatogonia (Sp), Sertoli cells (Se), spermatocytes (Sc), and spermatids (Sd), were evident. Lymphatic spaces between seminiferous tubules and adjacent to Leydig cells (L) clusters are clearly defined. After the slow rehydration-dehydration stepwise replacement with ETOH, testis tubules appeared normal with histological architecture similar to the control group (Figure 4(b)). Although all types of spermatogenic cell were identified after slow rehydration-dehydration stepwise replacement, spermatogonia and Sertoli cell nuclei were more difficult to distinguish compared to control. The quality of testicular histology observed after slow Re-ETOH only (Figure 4(c)) was similar to that observed in the slow rehydration-dehydration stepwise replacement group. However, open spaces (black arrows) were observed within portions of seminiferous tubules in the slow Re-ETOH only group, and the seminiferous tubule basement membrane in this group often appeared wavy and thinner (yellow arrows) compared to the control (Figure 4(a)). The histologic quality was poor in the group treated by single step (rapid transition) change of solution (Figure 4(d)), as evidenced by a diffuse appearance of the tissue (not due to focus) and limited clarity of nuclear details in spermatogonia and Sertoli cells. In addition, the seminiferous tubule basement membrane was occasionally indistinct and some spermatid artifactual loss of residual bodies is also observed.

Bottom Line: Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging.Postfixation processing was also standardized for safe shipment back to our laboratory.Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Mail Stop 3050, 3901 Rainbow Boulevard, HLSIC 3098, Kansas City, KS 66160, USA.

ABSTRACT
Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Show MeSH