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 source(s). 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.
Kukucka, Mark A. 1993. Mechanisms by which hypoxia augments Leydig
cell viability and differentiated cell function in vitro. Thesis (Ph.
D.) -- Virginia Polytechnic Institute and State University, 1993.