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Mechanisms By Which Hypoxia Augments Leydig Cell Viability And Differentiated Cell Function In Vitro  


Abstract Category: Science
Course / Degree: PhD
Institution / University: Virginia Tech, Blacksburg, VA, United States
Published in: 1993


Dissertation Abstract / Summary:

The 1980s heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells. The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal sources. Since luteinizing hormone LH and ascorbate are known to augment the production of oxytocin in ovarian granulose cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture. Highly enriched populations of guinea pig Leydig cells were isolated using a method that employed enzymatic dissociation and Percoll gradient centrifugation.

Since ambient oxygen tensions are toxic to cultured Leydig cells leading to decreased steroidogenic capacity, the antioxidant defense system of isolated Leydig cells was discerned. Decreased levels of several antioxidants including superoxide dismutase, glutathione reductase, glucose-6-phosphate dehydrogenase and total glutathione were measured. Using the dichlorofluorescin DCF-DA assay, it was determined that isolated Leydig cells were capable of accumulating hydrogen peroxide H2O2. Leydig cells maintained in an atmosphere composed of 19% oxygen produced H2O2 at a faster rate than similar cells incubated at 3% oxygen. Using a polyclonal antibody Ab-based immunoaffinity column, oxytocin biosynthesis was monitored in Leydig cells incubated with a mildly stimulating dose 0.1 ng/ml of ovine LH for 24, 48 and 72 hours in the presence of increasing concentrations of sodium ascorbate 1- 500 mM under culture conditions of hypoxia and normoxia.

Following solid phase extraction and immunoaffinity purification, sample supernatants were analyzed for both testosterone and oxytocin content as measured by radioimmunoassay RIA and high performance liquid chromatography-electrochemical detection HPLC-ECD respectively. Hypoxic culture conditions and low 1-10 mM concentrations of sodium ascorbate augmented the production of oxytocin from Leydig cells in culture. Higher 50-500 mM levels of ascorbate and normoxic culture conditions suppressed both testosterone and oxytocin production in isolated Leydig cells. Because oxytocin synthesis was found to be cycloheximide-sensitive, we conclude that Leydig cells possess the biosynthetic machinery necessary to manufacture oxytocin. The isolated oxytocin peptide was purified by HPLC with fraction collection followed by polyclonal-Ab immunoaffinity column chromatography.

Comparison of the amino acid sequence of the isolated octapeptide with authentic oxytocin provides unequivocal evidence that Leydig cells synthesize oxytocin de novo. Considering the widespread use of vitamin C as a dietary supplement, the research reported yields valuable mechanistic information on the reproductive biologic role of vitamin C in gonadal steroid and peptide hormone metabolism.


Dissertation Keywords/Search Tags:
Leydig cells, oxytocin, ascorbate, vitamin C, antioxidant defense system

This Dissertation Abstract may be cited as follows:
Kukucka, Mark A. 1993. Mechanisms by which hypoxia augments Leydig cell viability and differentiated cell function in vitro. Dissertation (Ph.D.) -- Virginia Polytechnic Institute and State University.


Submission Details: Dissertation Abstract submitted by Mark A. Kukucka, MS, DVM, PhD from United States on 18-Aug-2011 02:22.
Abstract has been viewed 2790 times (since 7 Mar 2010).



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