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.
