T. C. Anand Kumar

Bioavailability of progesterone enhanced by intranasal spraying

The bioavailability of progesterone (P) in terms of area under time-concentration curve and maximal concentration in the serum and cerebrospinal fluid was studied in adult ovariectomized rhesus monkeys following the administration of P as a nasal spray, i.v. or i.m. injections, nasal or eye drops. The bioavailability of P in both the body fluids was found to be considerably higher following its being sprayed intranasally.

Serum levels of dihydrotestosterone in male rhesus monkeys estimated by a non-chromatographic radioimmunoassay method

Since the antiserum used to measure dihydrotestosterone (DHT) has considerable cross-reaction with testosterone (T), the Chromatographie separation of DHT from T is a pre-requisite to get precise estimates of DHT levels by radioimmunoassay (RIA) in biological fluids. To simplify this procedure a non-chromatographic method for the estimation of DHT is reported. The treatment of T with 0.5% aqueous solution of potassium permanganate (KMnO4) for 30 min, at room temperature, selectively destroys its cross-reactivity with antiserum raised against T-3(O-carboxy-methyl)-oxime-bovine serum albumin. Whereas, the thin layer Chromatographie mobility of DHT in benzene-ethyl acetate solvent system (2:1, vv) and its cross-reaction with T-antiserum remain unaffected (~70%) following treatment with KMnO4. Thus by treating the ether extracts of biological samples with KMnO4 the endogenous T is inactivated and a simple non-chromatographic method is validated for quantitating DHT by RIA. In this assay procedure the serial dilutions of pools of serum samples from adult rhesus monkeys show dose-response curves that are parallel to the DHT standard curve. The correlation coefficient of DHT estimates obtained after Chromatographie separation of DHT on sephadex LH-20 column, using cyclohexane-benzene-methanol (80:15:15, by vol.) solvent system, or following KMnO4 treatment of ethereal extracts of serum samples is 0.99. Using this method, the geometric mean serum levels of DHT. measured in 20 adult male rhesus monkeys, are significantly (P < 0.05) higher at night (5.38 nmol/l) than during the day (3.22 nmol/l).

Testosterone, cortisol, prolactin and bioactive luteinizing hormone in day and night samples of cerebrospinal fluid and serum of male rhesus monkeys

Testosterone (T), cortisol (C), prolactin (PRL) and bioactive luteinizing hormone (bLH) were found to be normal constituents of the cerebrospinal fluid (CSF) of all the 15 adult male rhesus monkeys studied. The CSF levels of the hormones showed a good correlation with their serum levels. The geometric mean values of circulating levels of T, PRL, bLH in all the animals studied were significantly lower in the samples of the two body fluids collected between 09.00 and 11.00 h as compared with those collected between 21.00 and 23.00 h. C levels were higher during the day as compared with the night samples. This marked difference between the day and night levels of the circulating hormones was not observed in a few individuals which suggests that the diurnal changes in circulating levels of these hormones may not occur as a rule in all rhesus monkeys. The serum:CSF ratios for C, PRL and bLH did not vary significantly between the day and night samples of the body fluids as they did for T. This suggests that T is poorly transferred from the blood to the CSF as compared with the other 3 hormones studied. The possible pathways by which the hormones are transferred into the CSF and the functional significance of their presence in the CSF are discussed.

Histochemical localization of cholinesterases in the neural tissue of the pineal in the rhesus monkey

The neural tissue of the monkey pineal contains both acetyl and butyryl cholinesterases. Acetylcholinesterase was localized in the cisternae of the nuclear membrane, rough endoplasmic reticulum, on the plasma membrane of the neurones, and on the axolemma of both non-myelinated and myelinated fibres. The enzyme was not found in the axosomatic or axo-dendritic synapses. It is therefore suggested that the pineal neurones have a cholinergic function rather than a cholinoceptive one.

Transfer of Steroidal Hormones from Blood to the Cerebrospinal Fluid in the Rhesus Monkey

Rhesus monkeys were injected intravenously with tritium-labeled testosterone (T), progesterone (P), 17 α -hydroxyprogesterone (17 OHP), norethynodrel (N) and mestranol (M) to determin

Steroid metabolism by monkey and human spermatozoa

Freshly ejaculated spermatozoa from monkey and human were washed and incubated with tritium labelled androgens or estradiol to study the pattern of spermatozoa steroid metabolism. When equal concentrations of steroid substrates were used for incubation, monkey and human spermatozoa showed very similar pattern of steroid conversion. Spermatozoa from both species converted testosterone mainly to androstenedione, but reverse conversion of androstenedione to testosterone was negligible. Estradiol-17 beta was converted mainly to estrone. The close similarity between the spermatozoa of monkey and men in their steroid metabolic pattern indicates that the rhesus monkey could be an useful animal model to study the effect of drugs on the metabolic pattern of human spermatozoa.

Day and night levels of hormones in male rhesus monkeys kept under controlled or constant environmental light

The marked differences between day and night levels of testosterone, cortisol, prolactin and bioactive luteinizing hormone observed in adult male rhesus monkeys maintained at 12 h light∶12 h dark schedule are not changed by exposing monkeys to constant illumination for a period of up to 15 days. These findings suggest that the photoperiodicity of environmental light may not be the principal determinant for the occurrence of the diurnal rhythms observed in the circulating levels of the 4 hormones studied.

Estimation of daily sperm production in rats and monkeys

Daily sperm production in rats and monkeys were estimated by extended histometric method. Individual seminiferous tubules were separated, fixed, processed and embedded. Semi-thin sections were stained with toluidine blue and the different cell types quantitated with Chalkley’s eye piece. The daily sperm production estimated was compared with that obtained by the homogenization method. The results indicate that daily sperm production obtained by both the methods were comparable. The present histometric method has an added advantage of requiring very small amount of biopsy material to give precise daily sperm production estimations.

Circadian and circannual changes in the testicular function of adult rhesus monkeys

The endocrine and gametogenic status of the testes were studied in 9 healthy adult rhesus monkeys of proven fertility throughout a one-year period. Testosterone levels were estimated by radioimmunoassay in blood samples collected at 4 h intervals over a 24 h period once a month. Semen samples and testicular biopsies were also examined once a month. A well-defined circandian rhythm was evident in serum levels of testosterone. The rhythmicity was less pronounced in February and September. The 24 h mean levels of serum testosterone were high between the months of August to March and low in the months of May to July. All animals did not uniformly respond to electro-ejaculation in April and May. Semen volume and total number of spermatozoa were maximal between September and March and least from April to August. Testicular biopsies indicated that all stages of spermatogenesis were evident between September and March and the spermatogenic activity was less evident between April and August. The contents of Sertoli cells showed a seasonal cyclicity; they were laden with lipid droplets during April to August when spermatogenesis was quiescent and vacuolated during September to March when spermatogenesis was active. These studies indicate that the testing of contraceptive drugs needs to be restricted to months of September to March in male rhesus monkeys otherwise, it is possible that the naturally occurring reproductive quiscence may be attributed to the effect of the drug being tested. The data accrued from the present studies also provide quantitative information on circulating levels of testosterone which could be used as a reference background while evaluating the contraceptive drug-effects in male rhesus monkeys.

Association of Tritium-Labelled Estradiol, Progesterone and 17α-Hydroxyprogesterone with the Proteins of the Serum and Cerebrospinal Fluid in the Rhesus Monkey

The electrophoretic profiles of the proteins in the serum and cerebrospinal fluid (CSF) of the rhesus monkey were studied by polyacrylamide gel electrophoresis. The association of tritium labelled estradiol (3H-E), progesterone (3H-P) and 17 alpha-hydroxyprogesterone (3H-17OHP) with these proteins was studied under in vitro and in vivo conditions. The CSF was found to contain, besides albumin, at least 4 globulins with similar electrophoretic mobilities as those found in the serum. The electrophoretic mobilities of these globulins were Ralb 0.4, 0.6, 0.8 and 0.9. The electrophoretic profiles of the monkey serum and CSF proteins were found to be similar to those described for human serum and CSF. 3H-E and 3H-P were respectively associated with serum globulins of Ralb 0.4 and 0.8 both in the in vitro and in vivo studies. The respective Ralb of these 2 globulins were similar to the testosterone-estradiol binding globulin and cortisol binding globulin in human serum. 3H-17OHP was found to be associated with several globulins under both in vitro and in vivo conditions. In marked contrast to the in vitro studies, radioactive products were found to be associated with albumin in vivo. It is presumed that these products may be steroidal conjugates derived from the systemic metabolism of the administered steroids. No radioactivity was associated with the CSF protein after in vitro incubation with any of the three 3H-steroids. In the in vivo studies, however, radioactivity was associated with such of the CSF proteins that were similar to those found in the serum and with which the administered steroids were associated.