Measurement of serum prolactin and the effect of ketamine anesthesia on serum prolactin levels in cynomolgus monkeys (Macaca fascicularis)

The effect of an anesthetic, ketamine, on the serum prolactin level was examined in wild-originating female cynomolgus monkeys (Macaca fascicularis) imported from South East Asia. Serum prolactin levels were measured by the homologous radioimmunoassay system which was developed for human prolactin. The validity was confirmed by using an extract of pituitary gland from a female cynomolgus monkey as well as serum and amniotic fluid from a pregnant monkey. Additionally, serum luteinizing hormone (LH) levels were determined by the radioreceptor assay system developed in our laboratory using Leydig cells collected from rat's testes as a receptor fraction. The experiment was repeated three times at one-month interval, using twenty animals that were divided into three groups consisting of 5, 7 and 8 monkeys each. In the first experiment, the first group was injected with physiological saline and the second and third groups were intramuscularly given ketamine at a dose level of 5 mg/kg B.W. and 15 mg/kg B.W., respectively. In the second experiment, the first and second groups were given ketamine at a dose of 5 mg/kg B.W. and of 15 mg/kg B.W., respectively, and the third group was served as control injected with saline. In the third experiment, the first and third groups were administered with 15 mg/kg and 5 mg/kg of ketamine and the second group was injected with saline. In short, all of the twenty monkeys received the three different treatments for two months. The serum prolactin level showed a marked increase after the administration of ketamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased production of 11beta-hydroxysteroid dehydrogenase type 2 in the kidney microsomes of squirrel monkeys (Saimiri spp.)

In squirrel monkeys (Saimiri spp.), cortisol circulates at levels much higher than those seen in man and other Old World primates, but squirrel monkeys exhibit no physiologic signs of the mineralocorticoid effects of cortisol. These observations suggest that squirrel monkeys have mechanisms for protection of the mineralocorticoid receptor (MR) from these high levels of cortisol. We previously showed that the serum cortisol to cortisone ratio in these animals is low relative to that in human serum, suggesting that production of the MR protective enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), is increased in squirrel monkeys. Here, we directly evaluate whether increased production of 11beta-HSD2, which inactivates cortisol to cortisone, is a mechanism for protection of MR. In vitro assays showed that 11beta-HSD2 activity in squirrel monkey kidney microsomes was 3 to 7 times higher than that seen in kidney microsomes from pig or rabbit. 11beta-HSD2 protein detected by Western blot analysis was 4 to 9 times greater in squirrel monkey microsomes than in pig or rabbit microsomes. Comparison of the effect of expression of either human or squirrel monkey 11beta-HSD2 on MR transactivation activity showed similar inhibition of MR response to cortisol by both enzymes, indicating that the intrinsic activities of the human and squirrel monkey enzymes are similar. These findings suggest that one mechanism by which squirrel monkeys protect the MR from activation by high cortisol levels in the kidney is by upregulation of 11beta-HSD2 activity through increased production of the enzyme.     

Postnatal changes of IgG and IgM levels in squirrel monkeys (Saimiri sciureus)

Serum IgG and IgM levels were measured in domestically bred squirrel monkeys (Saimiri sciureus) ranging in age from 0 days to 42 months, as well as in adult squirrel monkeys from the wild estimated to be 60 months or older. The results indicated that the transplacental transfer of IgG occurs in the squirrel monkey but the transferability is lower in the squirrel monkey than in the cynomolgus monkey. Immune response in the squirrel monkey occurs just after birth, as shown by IgM production.     

An immunoreactive peptide of the FSH involved in autoimmune infertility.

The purpose of this study was to identify autoantigens contained in human ovary extracts. Serum samples from 36 infertile women with anti-ovary antibodies as detected with an ELISA technique were tested in Western blot against human ovary extracts. A reactive protein with a molecular mass matching that of the FSH was detected in 34 cases. These serum samples also reacted strongly in Western blot and ELISA with purified FSH and, in immunofluorescence, with pituitary cells. Using the Pepscan approach, with overlapping peptides matching the amino acid sequence of the human FSH beta-chain, several immunoreactive regions were evidenced. The 78-93 amino acid sequence of the human FSH beta-chain appeared as one of the major epitopes. Synthetic peptides of this region were prepared and demonstrated to react with human serum samples from women with anti-ovary antibodies. These data demonstrate that FSH can be an autoantigen, recognized by autoantibodies associated with infertility.     

Effects of single and multiple injections of ketamine hydrochloride on serum hormone concentrations in male cynomolgus monkeys

Using simulated short- and long-term effect studies, we evaluated the effect of ketamine anesthesia on serum cortisol, testosterone, and immunoreactive luteinizing hormone (ILH) and bioactive LH (BioLH) concentrations in adult male cynomolgus monkeys. Cortisol, testosterone, and ILH were measured by use of radioimmunoassay, and BioLH was measured by use of a radioreceptor assay method. For the acute effect, the first group (eight monkeys) was given four successive intramuscular injections of ketamine (10, 5, 5, and 5 mg/kg of body weight at 0, 30, 60, and 110 min respectively). Blood samples were taken at 0, 15, 30, 45, 60, and 120 min. For the long-term effect, the second group (10 monkeys) was given a single injection of ketamine (10 mg/kg) once a week for 4 consecutive weeks. Blood samples were taken 5 to 10 min after each injection, then were used to determine the variation in hormone concentrations among the monkeys (inter-individual variation) and within each monkey (intra-individual variation). There were no statistically significant differences in serum cortisol, testosterone, ILH, and BioLH values between the first blood sample (before the ketamine injection) and sequential blood samples in monkeys of the first group. Although intra-individual variation in the hormones (i.e., hormonal change within each monkey) was not statistically significant, inter-individual variation (among the monkeys) was significantly (0.00001 < P < 0.033) different in monkeys of the second group. These results indicate that an adequate number of animals must be used to minimize animal-to-animal variability. Our results confirm that ketamine is a suitable anesthetic agent to immobilize male cynomolgus monkeys in experimental studies (short- and long-term studies) aimed at elucidating hormonal changes.     

Determination of lactogenic activity in the serum of the squirrel monkey (Saimiri boliviensis) using the Nb2 lymphoma bioassay

Determination of squirrel monkey prolactin by immunoassay has been hampered by the lack of antiserum specific to prolactin from this species. As an alternate method, we have investigated whether the Nb2 lymphoma bioassay could be adapted for routine measurement of the lactogenic activity of samples of squirrel monkey serum. The growth of the Nb2 cells is absolutely dependent on the presence of lactogens in the culture medium. The cells were maintained in Fisher's medium supplemented with 10% horse serum, 10% fetal calf serum (FCS), and 10^?4M β-mercaptoethanol. For each assay, the cells were plated at an initial density of 1 × 10⁵ cells/ml in 22-mm 12-well dishes in the above medium, but devoid of FCS. Serum samples were heated to 56°C for 20 minutes to abolish the unusually high cytolytic complement activity of squirrel monkey serum and were incubated for 72 hours with Nb2 cells at serial dilutions from 1/40 to 1/2,560. Growth curves were generated with pooled samples of squirrel monkey serum, and the level of lactogenic activity was estimated using a calibration growth curve generated with known concentrations of purified rhesus monkey prolactin standard. We have found that the Nb2 lymphoma bioassay provides a sensitive and adaptable means for determination of lactogenic activity in the serum of the squirrel monkey.     

Hyperprolactinemia in monkeys: Induction by an estrogen-progesterone synergy

To evaluate the synergistic effect of estrogens and progesterone on prolactin secretion, rhesus and cynomolgus monkeys in the early follicular phase received estradiol benzoate (100 μg/kg/day, sc) alone for 14 days, then in combination with progesterone (subcutaneous silastic capsule) for an additional 14 days. Blood was drawn daily by femoral venipuncture under ketamine hydrochloride anesthesia (15mg/kg). Similarly, this protocol for exogenous steroid treatment was employed in a monkey having a chronically indwelling (femoral insertion into the vena cava) cannula maintained by a vest and mobile tether apparatus; however, no anesthesia was used to obtain serum specimens. In addition, this assembly was applied to six monkeys to determine the acute effects of ketamine hydrochloride on prolactin secretion. Concentrations of prolactin, estradiol-17β, and progesterone in serum were determined by conventional radioimmunoassays. Under estrogen therapy alone, mean circulating prolactin levels declined from ~15 to < 5 ng/ml; in contrast, the addition of progesterone caused an abrupt serum prolactin elevation, ~8–12 fold. This estradiol-progesterone course led to sustained hyperprolactinemia in the chronically catheterized Monkey, whereas ketamine administration raised serum prolactin only briefly, the elevation lasting less than three hours after injection. These findings establish that an estrogen-progesterone synergy, separate from the transient effects of ketamine, Induced hyperprolactinemia in cycling monkeys having prevailing levels of estrogen and progesterone near those characteristic of late gestation, when sustained prolactin elevations are observed normally.     

Conversion of androgens to estrogens in the male squirrel monkey (saimiri sciureas)

Many New World primates such as the squirrel monkey have extraordinarily high plasma levels of steroid hormones including cortisol, testosterone, progesterone and vitamin D₃. While plasma estrogen levels in female squirrel monkeys apparently are approximately the same as those found in other species no information is available for males. The present results indicate that the plasma levels of estrone (E1), estradiol (E2), and E1 sulfate are approximately 10-fold higher than those found in men. Comparative studies of androgen metabolism in genital skin fibroblasts indicate that squirrel monkey cells have higher aromatase and lower 5--reductase activity than human cells. Estimation of aromatase activity by a radiometric assay indicates that the high plasma estrogens are derived by peripheral conversion from testicular and/or adrenal androgens.     

Cortisol metabolism in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis)

New World squirrel monkeys (Saimiri spp.) have high circulating cortisol levels but normal electrolytes and blood pressures. The goal of the present study was to gain insight into adaptive mechanisms used by Bolivian squirrel monkeys to minimize the effects of high cortisol on mineralocorticoid receptor (MR) activity and electrolyte and water balance. Aldosterone levels in serum from 10 squirrel monkeys were 17.7 +/- 3.4 ng/dl (normal range in humans, 4 to 31 ng/dl), suggesting that squirrel monkeys do not exhibit a compensatory increase in aldosterone. The squirrel monkey MR was cloned and expressed in COS-7 cells and found to have similar responsiveness to cortisol and aldosterone as human MR, suggesting that squirrel monkey MR is not inherently less responsive to cortisol. To determine whether altered metabolism of cortisol might contribute to MR protection in squirrel monkeys, serum and urinary cortisol and cortisone were measured, and a comprehensive urinary corticosteroid metabolite profile was performed in samples from anesthetized and awake squirrel monkeys. The levels of cortisone exceeded those of cortisol in serum and urine, suggesting increased peripheral 11beta-hydroxysteroid dehydrogenase 2 activity in squirrel monkeys. In addition, a significant fraction (approximately 20%) of total corticosteroids excreted in the urine of squirrel monkeys appeared as 6beta-hydroxycortisol, compared with that in man (1%). Therefore, changes in cortisol metabolism likely contribute to adaptive mechanisms used by Bolivian squirrel monkeys to minimize effects of high cortisol.     

Androgen resistance in squirrel monkeys (Saimiri spp.)

The goal of this study was to understand the basis for high androgen levels in squirrel monkeys (Saimiri spp.). Mass spectrometry was used to analyze serum testosterone, androstenedione, and dihydrotestosterone of male squirrel monkeys during the nonbreeding (n = 7) and breeding (n = 10) seasons. All hormone levels were elevated compared with those of humans, even during the nonbreeding season; the highest levels occurred during the breeding season. The ratio of testosterone to dihydrotestosterone in squirrel monkeys is high during the breeding season compared to man. Squirrel monkeys may have high testosterone to compensate for inefficient metabolism to dihydrotestosterone. We also investigated whether squirrel monkeys have high androgens to compensate for low-activity androgen receptors (AR). The response to dihydrotestosterone in squirrel monkey cells transfected with AR and AR-responsive reporter plasmids was 4-fold, compared with 28-fold in human cells. This result was not due to overexpression of cellular FKBP51, which causes glucocorticoid and progestin resistance in squirrel monkeys, because overexpression of FKBP51 had no effect on dihydrotestosterone-stimulated reporter activity in a human fibroblast cell line. To test whether the inherently low levels of FKBP52 in squirrel monkeys contribute to androgen insensitivity, squirrel monkey cells were transfected with an AR expression plasmid, an AR-responsive reporter plasmid, and a plasmid expressing FKBP52. Expression of FKBP52 decreased the EC50 or increased the maximal response to dihydrotestosterone. Therefore, the high androgen levels in squirrel monkeys likely compensate for their relatively low 5 alpha-reductase activity during the breeding season and AR insensitivity resulting from low cellular levels of FKBP52.     

Novel hyperprolactinemia and hyperprolactinemic anovulation model using the cynomolgus monkey (Macaca fascicularis)

We investigated the relationship between the menstrual cycle and hormone levels in cynomolgus monkeys, and developed a sulpiride-induced hyperprolactinemic anovulation model. On this study, we demonstrated the usefulness of the commercial human prolactin immunoradiometric assay kit for the measurement of cynomolgus monkey serum samples. In the normal menstrual cycle of the cynomolgus monkey, serum prolactin concentrations were not significantly different between luteal and follicular phases. However, the serum prolactin concentration tended to elevate at the ovulation stage. And serum progesterone began to increase after an estradiol surge, and then declined before the ensuing preovulatory rise in estradiol. During the luteal phase, the serum concentration of progesterone was elevated. Moreover, we aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in the cynomolgus monkey. The serum prolactin level gradually increased during the twice-daily administration of sulpiride, and the drug produced as big a response at 5 mg/kg. In this study, the length of the menstrual cycle was approximately 29 days in normal cynomolgus monkeys. When treatment with sulpiride had been continued for more than one month, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle, and the absence of ovulation was recognized by laparoscopy. Moreover, in this period we found that amenorrhea or anovulatory menstruation in the experimental animals. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. Our findings suggest that the cynomolgus monkey is useful as a endocrinological model that uses prolactin as a parameter and as an anovulatory model; thus, it could be a useful model for the hyperprolactinemic amenorrhea and/or anovulation seen in humans.     

Purification of monkey prolactin from culture medium: biochemical and immunological characterization

Serum-free culture medium, previously incubated with dispersed monkey pituitary cells, provided a relatively uncontaminated source for extraction and purification of 15 mg of monkey prolactin. N-terminal group analysis of this preparation, M21GB, produced predominantly leucine. The amino acid composition closely resembles that of both human and sheep prolactin. M21GB monkey prolactin migrates in 10% polyacrylamide with sodium dodecyl sulfate (SDS) as a single band with a molecular weight of about 23,000. Multiple bands typical of prolactins are seen with nondenaturing polyacrylamide disc electrophoresis. M21GB contains less than 1% growth hormone, incorporates radioactive iodine with a specific activity of 15 microCi/micrograms and specifically binds to the anti-human prolactin serum-3 provided by the National Hormone and Pituitary Program (B spec/total = 23%). M21GB does not compete in a linear fashion with iodinated human prolactin-16 for the human prolactin antiserum, but M21GB does compete in a linear fashion with iodinated M21GB with the same antiserum. Monkey serum, pituitary homogenate, and culture medium containing unknown levels of monkey prolactin are not parallel with NIAMDD-HPrl-RP1 in the human prolactin assay, but are parallel when M21GB is used as the reference preparation and for iodination. Finally, antisera to M21GB were generated in rabbits which are specific for monkey and human prolactin and which can be used for radioimmunoassay or immunocytochemistry. In summary, serum-free medium from primary cultures of dispersed monkey pituitaries provided a quantitity of monkey prolactin which promoted biochemical analysis and production of a specific antiserum. This culture system may be a unique and ongoing source for extraction of significant quantities of monkey prolactin suitable for investigative use.     

Immunofluorescence study of the hypothalamo-infundibular LRH tract and serum gonadotropin levels in the female squirrel monkey during the estrous cycle

Summary The study of LRH reactive neurons of the medial basal hypothalamic group was made with rabbit antisera to unconjugated synthetic LRH, in normally cycling female squirrel monkeys.The specifically immunoreactive material present along the hypothalamo-infundibular LRH tract shows significant modifications that are particularly distinct in the lateral, posterior and anterior labia of median eminence, with a maximum concentration during the early and middle follicular phases, a sudden fall to a minimum concentration during the late follicular and ovulatory phases and with a progressive increase during the luteal and early follicular phases.Serum FSH and LH levels show a progressive decrease from the luteal phase to the late follicular phase, with differential modifications suggesting that a double regulation might take place in cycling squirrel monkeys.A parallel is suggested between the specifically reactive material along the hypothalamo-infundibular tract, serum gonadotropin levels and variations in the medial basal hypothalamic and infundibular LRH concentrations during the estrous cycle in the squirrel monkey.This work was supported by the D.G.R.S.T. (contrat 76-7-1536) and U.E.R. 3 (Faculty of Medicine). We acknowledge the help of A. Pillez (C.N.R.S.) for sectioning and staining the genital tracts     

Monoclonal Antibodies Specific for Members of the Genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni , M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas. Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni , M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64–200 kD.     

Monoclonal antibodies specific for members of the genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni, M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas, Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni, M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64-200 kD.     

Studies on ovarian quiescence in the lactating bonnet monkey (Macaca radiata)

Lactating bonnet monkeys were used as a model to understand the mechanism of ovarian quiescence during lactation. The ovary of the bonnet monkey in the 3rd month of lactation responds well to exogenous pregnant mare serum gonadotropin stimulation with serum estrogen values reaching maximal levels by day 3 of the gonadotropin injection. The adminstration of ovine prolactin to such monkeys significantly inhibited the ovarian responsiveness to exogenous gonadotropin. The responsiveness of the pituitary of the lactating monkey (in the 3rd month of lactation) to luteinizing hormone releasing hormone injection was suppressed and supplementation with exogenous prolactin further accentuating this effect. The relative ability of chlorpromazine given intravenously/intramuscularly/intranasally to enhance endogenous prolactin levels was assessed. During the first 5 months of lactation when the basal prolactin levels were high, the luteinizing hormone levels were low. As the suckling stimulus reduces and prolactin levels fall, luteinizing hormone levels increase, the first post-parturient mensus occurring by 218 ± 4 days. This event was postponed by 3 months on increasing endogenous prolactin levels by administering chlorpromazine (250 μg/day by intranasal mode) over a 5 day period every month starting from the 3rd month of lactation.     

Immobilization with a single dose of ketamine hydrochloride and a combination of xylazine hydrochloride-ketamine hydrochloride and antagonism by yohimbine hydrochloride in the Japanese monkey (Macaca fuscata)

Forty-nine free-ranging Japanese monkeys (Macaca fuscata) were immobilized with 4.3–15.6 mg/kg (mean±S.D.=10.0±2.5 mg/kg) of ketamine hydrochloride (HCl), and 27 Japanese monkeys kept in enclosures were immobilized with a combination of 0.8–1.4 mg/kg (1.0±0.2 mg/kg) of xylazine HCl and 4.0–7.1 mg/kg (5.0±0.6 mg/kg) of ketamine HCl. In the xylazine HCl-ketamine HCl combination, good myorelaxation was induced. The mean induction times for the single dosage of ketamine HCl and the xylazine HCl-ketamine HCl combination were 2.8±1.5 min and 6.9±4.4 min, respectively. The mean immobilization times with the single dosage of ketamine HCl and the xylazine HCl-ketamine HCl combination were 39.3±16.5 min and 58.8±34.2 min, respectively. A half dose of ketamine HCl in combination with xylazine HCl could also immobilize Japanese monkeys successfully. Administrations of 0.5 mg/kg i.v. and 1.0 mg/kg i.m. of yohimbine HCl as an antagonist to xylazine HCl at 30 min after the induction reduced the immobilization time to 31.4±0.5 min and 49.0±22.1 min, respectively. Yohimbine HCl appears to be an effective antagonist to combination anesthesia by xylazine HCl-ketamine HCl in the Japanese monkey.