A specific high-performance liquid chromatographic (HPLC) method is described for the reliable quantitation of oxytocin using culture media supernatants. The procedure employs solid-phase extraction, antibody-based immunoaffinity purification and isocratic HPLC with dual channel coulometric detection (ED). The lower limit of detection for this cyclic nonapeptide was 40 pg (40 fmol). Due to its relative simplicity, specificity and precision, the HPLC-ED of oxytocin is an accurate and attractive alternative to many existing quantitative methods.

In recent years, several metabolic roles have been proposed for vitamin C. Recent information suggests a strong causal relationship between high endogenous levels of ascorbic acid and changes in normal reproductive biology. Using highly enriched populations of guinea pig Leydig cells, we have found that elevated levels (50 to 500 microM) of ascorbate significantly (P < 0.01) depressed testosterone production in a dose-dependent manner while low levels (0 to 10 microM) were without effect. Leydig cells incubated under hypoxic (3% oxygen) culture conditions produced significantly (P < 0.01) more testosterone than similar cells cultured under normoxic (19% oxygen) conditions. The results of this study suggest that high concentrations of ascorbate and normoxic culture conditions suppress testosterone production in isolated Leydig cells. Thus, it would seem that there exists a delicate balance between normal metabolic requirements for vitamin C and excessive ascorbate levels that might alter normal gonadal reproductive events.

Leydig cells were isolated from adult male guinea-pig testes using a multi-step procedure involving enzymatic dissociation and Percoll-gradient centrifugation. The following description is the first account of a successful isolation of adolescent guinea-pig Leydig cells. The enriched Leydig-cell preparation routinely isolated from six intact testicles yielded approximately 5.0 x 10(6) +/- 0.7 x 10(6) (+/- SEM) Leydig cells with a viability of 98.0 +/- 0.4% as determined using the trypan-blue exclusion method. The purity of the isolated cell population as assessed by 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) staining averaged 82.5 +/- 0.8%. Under light microscopy, guinea-pig Leydig cells were polyhedral in shape with a large prominent nucleus and a distinct nucleolus. The acidophilic cytoplasm contained numerous lipid-filled vesicles. Ultrastructurally, guinea-pig Leydig cells displayed an eccentrically located ovoid nucleus with dark-staining peripheral heterochromatin. Large quantities of mitochondria, smooth endoplasmic reticulum and particulate-laden lipid droplets were also evident. The steroidogenic potential of the isolated Leydig cells was verified using a maximally stimulating dose of ovine LH (100 ng ml-1) and human CG (200 mIU ml-1). Leydig cells incubated in a shaking (120 cycles min-1) water bath for 3 h at 37 degrees C in capped polypropylene microcentrifuge tubes produced 233 +/- 21 ng and 223 +/- 18 ng testosterone per 1 x 10(6) cells when maximally stimulated with oLH or hCG, respectively. The inclusion of low (1-5 microM) levels of sodium ascorbate during culture enhanced significantly Leydig-cell viability vs. control values.

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.

Utilization of highly enriched preparations of steroidogenic Leydig cells have proven invaluable for studying the direct effects of various hormones and agents on Leydig cell function in vitro. However, recent work indicates that isolated Leydig cells are often subjected to oxygen (O2) toxicity when cultured at ambient (19%) oxygen concentrations. Because intracellular antioxidants play an important role in protecting cells against oxygen toxicity, we have investigated the intracellular antioxidant defense system of isolated Leydig cells. The cellular levels of several antioxidants including catalase, glucose-6-phosphate dehydrogenase (G-6-PDH), superoxide dismutase (SOD) of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione reductase and total glutathione were quantitated using enriched populations of Leydig cells isolated from adult male guinea pig testes. Compared to whole testicular homogenates, Leydig cells contained significantly (P &lt; 0.01) less G-6-PDH, total SOD, glutathione reductase and total glutathione, but significantly (P &lt; 0.001) more glutathione peroxidase. Compared to hepatic values previously reported in the guinea pig, Leydig cells contain nearly 400 times less catalase, about 14 times less glutathione peroxidase and almost 11 times less glutathione reductase. Since G-6-PDH and glutathione reductase are both necessary to regenerate reduced glutathione (GSH) which couples with glutathione peroxidase to breakdown hydrogen peroxide (H2O2) under normal conditions, it is plausible that the oxygen toxicity observed in isolated Leydig cells is due to the intracellular accumulation of H2O2. Using the dichlorofluorescin diacetate (DCF-DA) assay, we found that Leydig cells incubated in the presence of 19% O2 produced significantly (P<0.001) higher levels of H2O2 with time in culture compared to Leydig cells maintained at 3% O2. These results support the hypothesis that the increased susceptibility of isolated Leydig cells to oxygen toxicity may be due, in part, to decreased amounts of certain antioxidant defenses and an increased production of the reactive oxygen species H2O2.

Highly purified populations of guinea pig Leydig cells were incubated with a maximally stimulating dose of 100 ng/mL LH for 24 h in the presence of increasing concentrations of sodium ascorbate. Sample supernatants were extracted, concentrated under vacuum, and reconstituted with acidified absolute ethanol. Samples were analyzed for oxytocin using high-performance liquid chromatography with electrochemical detection and known concentrations of an authentic oxytocin standard. Leydig cells stimulated with 0, 25, and 50 microM ascorbate produced and secreted 40.1 +/- 1.23, 77.4 +/- 13.8, 74.2 +/- 26.3 pg of an oxytocin-like peptide, respectively, per 1 x 10(6) cells. These results indicate that guinea pig Leydig cells are capable of producing an oxytocin-like peptide de novo and that low concentrations of ascorbate stimulate the production of this peptide in Leydig cells cultured in vitro.

The neurohypophysial peptide hormone oxytocin is associated with parturition/milk ejection. The identification of a non-neural source of oxytocin secreted in significant amounts from the ovarian corpus luteum of ruminants stimulated interest in this octapeptide hormone. Oxytocin has an ovarian source, whereas an oxytocin-like peptide is reportedly present in the testes. Highly purified populations of guinea pig Leydig cells were incubated with a maximally stimulating dose of 100 ng/ml LH for 24 hours in the presence of increasing concentrations of sodium ascorbate. Sample supernatants were extracted, concentrated under vacuum and reconstituted with acidified absolute ethanol. Samples were analyzed for oxytocin using high performance liquid chromatography (HPLC) with electrochemical detection and known concentrations of an authentic oxytocin standard. Leydig cells stimulated with 0, 25 and 50 uM ascorbate produced and secreted 40 +/- 1.2, 77 +/- 14 and 74 +/- 26 pg of an oxytocin-like peptide, respectively, per 1 x 10(6) cells. It would appear that guinea pig Leydig cells are capable of producing an oxytocin-like peptide de novo. Low concentrations of ascorbate stimulate the production of this peptide in Leydig cells cultured in vitro. Comparison of the amino acid composition and sequence of this oxytocin-like peptide with authentic oxytocin are already in progress to confirm whether Leydig cells possess the biosynthetic machinery for testicular oxytocin production.

Efficient use of commercial rabbit production facilities include housing the optimum number of rabbits per unit of area without adversely affecting the performance or the health of the animals. An experiment was conducted to determine the effect of pen design on animal production and management. Four different pen designs of equal area (0.74 sq. m) but of unequal perimeter were employed to ascertain the spacing relationships of domestic rabbits housed eight to a pen. Pen types were: 1) square pen, 2) square pen with four partitions (total length 1.7 m), 3) square pen with raised platform (0.10 m wide and 0.25 m high) attached to the interior walls of the pen, and 4) an equilateral triangular pen. Spatial and behavioral parameters were monitored one day each week for four weeks using time-lapse cameras. Weekly measurements of individual rabbit body weights were recorded. Individual head coordinates were digitized from the photographs. From the head coordinates, 3-dimensional surface plots were drawn of the sum of usage of each portion of each pen for the activity recorded weekly. The surface plots indicated there was variation among pen types and over weeks in spatial patterns. The locations most preferred by the rabbits could be determined from the 3-dimensional graphs. Distance to group center and distance to pen center were greatest for the platform pen, intermediate for the triangle pen and smallest for the partitioned and conventional pens. Female rabbits were heavier and rested more than male rabbits. The behavioral variables crouching and resting were highly correlated with initial weight, final weight and average daily gain. Rabbits that crouched more than they rested gained less weight. The percentage of time spent on the platform was 19, 23, 30 and 36 from weeks 1 through 4, respectively. The results suggest that the platform pen may be an effective means of reducing animal crowding by increasing the amount of space available to a group of animals.