FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / In Vitro Cell Dev Biol Anim / In vitro culture of precision-cut testicular tissue as a nov...
In vitro cellular & developmental biology. Animal2009; 46(1); 45-53; doi: 10.1007/s11626-009-9242-1

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH.

Abstract: In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n=3) and cut into slices (mean slice weight= 13.85 ± 0.20 mg; mean slice thickness=515.00 ± 2.33 μm) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50, and 500 ng/ml for 6 h at 32 °C. Viability of the tissue was maintained based on histological integrity and lack of appreciable lactate dehydrogenase in the medium. The production and release of testosterone (T) and estradiol-17β (E2) into the medium was measured following in vitro culture. The addition of oLH increased T and E2 at least 400% and 120%, respectively, over the 0-ng oLH control cultures. Testicular gene expression was assessed with in situ hybridization methodology for steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs. In situ hybridization revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA in Leydig cells. No differences were observed for the expression of PDE3B or ODF2 genes in seminiferous tubules among treatment groups as expected. These results demonstrate the value of in vitro culture of the precision-cut tissue slices for studies of the regulation of steroidogenesis and gene expression in the stallion testes.
Get Full Text
Publication Date: 2009-11-17 PubMed ID: 19915939DOI: 10.1007/s11626-009-9242-1Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article discusses how precision-cut in vitro cultures of testicular tissue can be used to study responses to luteinizing hormone (LH) in stallions. The tissue cultures maintained viability and revealed significant increases in testosterone and estradiol production alongside the concentration-dependent increase of StAR protein mRNA in response to the LH.

Research Procedure

  • The study begins with the collection of testes from light breed stallions that are immature. They are then cut into slices by using a precision cut tissue slicing method.
  • Four slices of this tissue are placed on a grid that is floating on a medium within individual vials.
  • After a preincubation period that lasts an hour, they are subjected to exposure to a medium that contains oLH (ovine luteinizing hormone). The oLH concentrations used in this study are 0, 5, 50, and 500 ng/ml, and exposure is for 6 hours at 32 °C.

Assessment of Tissue Viability

  • The researchers determined the viability of these tissue slices by assessing histological integrity and observing for the absence of significant lactate dehydrogenase levels in the medium. This enzyme typically indicates cell damage or death.
  • The production and release of both testosterone (T) and estradiol-17β (E2) into the medium were also measured following the in vitro culture, which further confirms tissue viability.

Effects of Luteinizing Hormone Exposure

  • The research found that exposing the tissues to oLH increased T and E2 production by at least 400% and 120%, respectively, over cultures that didn’t receive oLH exposure.
  • The researchers also assessed the expression of steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs through in situ hybridization.
  • The in situ process revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA found in Leydig cells (cells that produce testosterone in mammals).
  • No differences were registered for the expression of PDE3B or ODF2 genes in seminiferous tubules among the different treatment groups, which was an expected outcome.

Conclusion of the Study

  • The research shows the value of precision-cut in vitro cultures for studying the regulation of steroidogenesis and gene expression within the testes of stallions.
  • This novel method serves as a valuable tool for future studies on reproductive mechanisms in animals or for biomedical applications.

Cite This Article

APA
Laughlin AM, Welsh TH, Love CC, Varner DD, Parrish AR, Forrest DW, Ing NH. (2009). In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH. In Vitro Cell Dev Biol Anim, 46(1), 45-53. https://doi.org/10.1007/s11626-009-9242-1

Publication

In vitro cellular & developmental biology. Animal
ISSN: 1543-706X
NlmUniqueID: 9418515
Country: Germany
Language: English
Volume: 46
Issue: 1
Pages: 45-53

Researcher Affiliations

Laughlin, Andy Michael
  • Department of Animal Science, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX 77843, USA.
Welsh, Thomas H
    Love, Charles C
      Varner, Dickson D
        Parrish, Alan R
          Forrest, David W
            Ing, Nancy H

              MeSH Terms

              • Animals
              • Cell Shape / drug effects
              • Cyclic Nucleotide Phosphodiesterases, Type 3 / genetics
              • Cyclic Nucleotide Phosphodiesterases, Type 3 / metabolism
              • Estradiol / biosynthesis
              • Gene Expression Regulation / drug effects
              • Heat-Shock Proteins / genetics
              • Heat-Shock Proteins / metabolism
              • Horses
              • In Situ Hybridization
              • L-Lactate Dehydrogenase / metabolism
              • Luteinizing Hormone / pharmacology
              • Male
              • Phosphoproteins / genetics
              • Phosphoproteins / metabolism
              • RNA, Messenger / genetics
              • RNA, Messenger / metabolism
              • Sheep
              • Testis / cytology
              • Testis / drug effects
              • Testis / enzymology
              • Testosterone / biosynthesis
              • Tissue Culture Techniques / methods

              References

              This article includes 26 references
              1. Steinberger A, Klinefelter G. Sensitivity of Sertoli and Leydig cells to xenobiotics in in vitro models.. Reprod Toxicol 1993;7 Suppl 1:23-37.
                pubmed: 8400637doi: 10.1016/0890-6238(93)90066-ggoogle scholar: lookup
              2. Ing NH, Spencer TE, Bazer FW. Estrogen enhances endometrial estrogen receptor gene expression by a posttranscriptional mechanism in the ovariectomized ewe.. Biol Reprod 1996 Mar;54(3):591-9.
                pubmed: 8835380doi: 10.1095/biolreprod54.3.591google scholar: lookup
              3. Parks JE, Lee DR, Huang S, Kaproth MT. Prospects for spermatogenesis in vitro.. Theriogenology 2003 Jan 1;59(1):73-86.
                pubmed: 12499019doi: 10.1016/s0093-691x(02)01275-xgoogle scholar: lookup
              4. Manna PR, Stocco DM. Regulation of the steroidogenic acute regulatory protein expression: functional and physiological consequences.. Curr Drug Targets Immune Endocr Metabol Disord 2005 Mar;5(1):93-108.
                pubmed: 15777208doi: 10.2174/1568008053174714google scholar: lookup
              5. Luo L, Chen H, Stocco DM, Zirkin BR. Leydig cell protein synthesis and steroidogenesis in response to acute stimulation by luteinizing hormone in rats.. Biol Reprod 1998 Aug;59(2):263-70.
                pubmed: 9687294doi: 10.1095/biolreprod59.2.263google scholar: lookup
              6. Eisenhauer KM, Roser JF. Effects of lipoprotein, equine luteinizing hormone, equine follicle-stimulating hormone, and equine prolactin on equine testicular steroidogenesis in vitro.. J Androl 1995 Jan-Feb;16(1):18-27.
                pubmed: 7768749
              7. Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity.. J Immunol Methods 1988 Nov 25;115(1):61-9.
                pubmed: 3192948doi: 10.1016/0022-1759(88)90310-9google scholar: lookup
              8. Ing NH, Tornesi MB. Estradiol up-regulates estrogen receptor and progesterone receptor gene expression in specific ovine uterine cells.. Biol Reprod 1997 May;56(5):1205-15.
                pubmed: 9160720doi: 10.1095/biolreprod56.5.1205google scholar: lookup
              9. Hess MF, Roser JF. A comparison of the effects of equine luteinizing hormone (eLH), equine growth hormone (eGH) and human recombinant insulin-like growth factor (hrIGF-I) on steroid production in cultured equine Leydig cells during sexual maturation.. Anim Reprod Sci 2005 Oct;89(1-4):7-19.
                pubmed: 16085376doi: 10.1016/j.anireprosci.2005.06.014google scholar: lookup
              10. Parrish AR, Gandolfi AJ, Brendel K. Precision-cut tissue slices: applications in pharmacology and toxicology.. Life Sci 1995;57(21):1887-901.
                pubmed: 7475939doi: 10.1016/0024-3205(95)02176-jgoogle scholar: lookup
              11. Azri-Meehan S, Mata HP, Gandolfi AJ, Brendel K. The hepatotoxicity of chloroform in precision-cut rat liver slices.. Toxicology 1992;73(3):239-50.
                pubmed: 1631901doi: 10.1016/0300-483x(92)90066-ngoogle scholar: lookup
              12. Staub C. A century of research on mammalian male germ cell meiotic differentiation in vitro.. J Androl 2001 Nov-Dec;22(6):911-26.
                pubmed: 11700854doi: 10.1002/j.1939-4640.2001.tb03430.xgoogle scholar: lookup
              13. Smith PF, Gandolfi AJ, Krumdieck CL, Putnam CW, Zukoski CF 3rd, Davis WM, Brendel K. Dynamic organ culture of precision liver slices for in vitro toxicology.. Life Sci 1985 Apr 8;36(14):1367-75.
                pubmed: 3982217doi: 10.1016/0024-3205(85)90042-6google scholar: lookup
              14. Manna PR, Huhtaniemi IT, Stocco DM. Detection of hCG Responsive Expression of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells.. Biol Proced Online 2004;6:83-93.
                pubmed: 15181477doi: 10.1251/bpo76google scholar: lookup
              15. Catania JM, Parrish AR, Kirkpatrick DS, Chitkara M, Bowden GT, Henderson CJ, Wolf CR, Clark AJ, Brendel K, Fisher RL, Gandolfi AJ. Precision-cut tissue slices from transgenic mice as an in vitro toxicology system.. Toxicol In Vitro 2003 Apr;17(2):201-5.
                pubmed: 12650674doi: 10.1016/s0887-2333(03)00004-3google scholar: lookup
              16. Clark BJ, Wells J, King SR, Stocco DM. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR).. J Biol Chem 1994 Nov 11;269(45):28314-22.
                pubmed: 7961770
              17. Boitani C, Politi MG, Menna T. Spermatogonial cell proliferation in organ culture of immature rat testis.. Biol Reprod 1993 Apr;48(4):761-7.
                pubmed: 8485240doi: 10.1095/biolreprod48.4.761google scholar: lookup
              18. Parrish AR, Sallam K, Nyman DW, Orozco J, Cress AE, Dalkin BL, Nagle RB, Gandolfi AJ. Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology.. Cell Biol Toxicol 2002;18(3):205-19.
                pubmed: 12083426doi: 10.1023/a:1015567805460google scholar: lookup
              19. Ing NH, Laughlin AM, Varner DD, Welsh TH Jr, Forrest DW, Blanchard TL, Johnson L. Gene expression in the spermatogenically inactive "dark" and maturing "light" testicular tissues of the prepubertal colt.. J Androl 2004 Jul-Aug;25(4):535-44.
                pubmed: 15223842doi: 10.1002/j.1939-4640.2004.tb02824.xgoogle scholar: lookup
              20. Webber KM, Stocco DM, Casadesus G, Bowen RL, Atwood CS, Previll LA, Harris PL, Zhu X, Perry G, Smith MA. Steroidogenic acute regulatory protein (StAR): evidence of gonadotropin-induced steroidogenesis in Alzheimer disease.. Mol Neurodegener 2006 Oct 3;1:14.
                pubmed: 17018137doi: 10.1186/1750-1326-1-14google scholar: lookup
              21. Stocco DM, Clark BJ. Regulation of the acute production of steroids in steroidogenic cells.. Endocr Rev 1996 Jun;17(3):221-44.
                pubmed: 8771357doi: 10.1210/edrv-17-3-221google scholar: lookup
              22. Parrish AR, Zheng XH, Turney KD, Younis HS, Gandolfi AJ. Enhanced transcription factor DNA binding and gene expression induced by arsenite or arsenate in renal slices.. Toxicol Sci 1999 Jul;50(1):98-105.
                pubmed: 10445758doi: 10.1093/toxsci/50.1.98google scholar: lookup
              23. Orth JM, McGuinness MP, Qiu J, Jester WF Jr, Li LH. Use of in vitro systems to study male germ cell development in neonatal rats.. Theriogenology 1998 Jan 15;49(2):431-9.
                pubmed: 10732024doi: 10.1016/s0093-691x(97)00415-9google scholar: lookup
              24. Schmelz M, Schmid VJ, Parrish AR. Selective disruption of cadherin/catenin complexes by oxidative stress in precision-cut mouse liver slices.. Toxicol Sci 2001 Jun;61(2):389-94.
                pubmed: 11353148doi: 10.1093/toxsci/61.2.389google scholar: lookup
              25. Parrish AR, Catania JM, Orozco J, Gandolfi AJ. Chemically induced oxidative stress disrupts the E-cadherin/catenin cell adhesion complex.. Toxicol Sci 1999 Sep;51(1):80-6.
                pubmed: 10496679doi: 10.1093/toxsci/51.1.80google scholar: lookup
              26. Pollack SE, Furth EE, Kallen CB, Arakane F, Kiriakidou M, Kozarsky KF, Strauss JF 3rd. Localization of the steroidogenic acute regulatory protein in human tissues.. J Clin Endocrinol Metab 1997 Dec;82(12):4243-51.
                pubmed: 9398748doi: 10.1210/jcem.82.12.4445google scholar: lookup

              Citations

              This article has been cited 4 times.
              1. Meijer TG, Naipal KA, Jager A, van Gent DC. Ex vivo tumor culture systems for functional drug testing and therapy response prediction. Future Sci OA 2017 Jun;3(2):FSO190.
                doi: 10.4155/fsoa-2017-0003pubmed: 28670477google scholar: lookup
              2. Tang L, Chai W, Ye F, Yu Y, Cao L, Yang M, Xie M, Yang L. HMGB1 promotes differentiation syndrome by inducing hyperinflammation via MEK/ERK signaling in acute promyelocytic leukemia cells. Oncotarget 2017 Apr 18;8(16):27314-27327.
                doi: 10.18632/oncotarget.15432pubmed: 28404891google scholar: lookup
              3. Zhou PH, Hu W, Zhang XB, Wang W, Zhang LJ. Protective Effect of Adrenomedullin on Rat Leydig Cells from Lipopolysaccharide-Induced Inflammation and Apoptosis via the PI3K/Akt Signaling Pathway ADM on Rat Leydig Cells from Inflammation and Apoptosis. Mediators Inflamm 2016;2016:7201549.
                doi: 10.1155/2016/7201549pubmed: 27212810google scholar: lookup
              4. Amato CM, Yao HH. New uses for an old technique: live imaging on the slice organ culture to study reproductive processes†. Biol Reprod 2024 Jun 12;110(6):1055-1064.
                doi: 10.1093/biolre/ioae023pubmed: 38315794google scholar: lookup
              Subscribe to our newsletter
              Join 99,383 horse owners like you who receive our email updates on horse health and nutrition.