Leptin and thyroxine during sexual development in male monkeys: effect of neonatal gonadotropin-releasing hormone antagonist treatment and delayed puberty on the developmental pattern of leptin and thyroxine secretion.

OBJECTIVE: Neonatal treatment of male monkeys with a gonadotropin-releasing hormone antagonist (Ant) increased the incidence of delayed puberty. Using blood samples that had been collected from monkeys with normal or delayed puberty, we assessed the potential involvement of leptin and thyroxine (T4) in sexual development. DESIGN AND METHODS: Monkeys were treated from birth until 4 months of age with vehicle, Ant or Ant/androgen and blood samples were drawn from 10 to 62 months of age. RESULTS: Serum leptin and total T4 concentrations declined in parallel throughout adolescence in all treatment groups. There was no transient rise in leptin before or in association with the onset of puberty. Also, leptin did not differ during the peripubertal period between animals experiencing puberty at that time versus those in which puberty was being delayed. Neonates treated with Ant either alone or with androgen replacement had higher leptin levels than controls throughout development. While leptin exhibited no significant changes during the peripubertal period, T4 values increased and declined in parallel with the peripubertal changes in hypothalamic-pituitary-testicular activity. CONCLUSIONS: These data do not support the concept that a transient rise in leptin triggers the onset of puberty in male monkeys. However, the disruption of neonatal activity of the pituitary-testicular axis alters the developmental pattern of leptin. The changes in T4 levels during the peripubertal period suggest that thyroid status may be a significant contributor to the process of sexual development in the male monkey and that peripubertal changes in secretion of this hormone may serve as an effective physiological response during a critical period of elevated energy expenditure.

Endocrine-immune interaction: alteractions in immune function resulting from neonatal treatment with a GnRH antagonist and seasonality in male primates.

PROBLEM: The effect of neonatal gonadotropin releasing hormone (GnRH) antagonist (Ant) treatment and seasonality on immune system development and function was investigated in male primates. METHOD OF STUDY: Neonatal male rhesus monkeys and marmosets were treated with Ant, and its effect on immune system morphology, circulating lymphocyte subsets, and cell- and humorally-mediated immune responses was assessed during development. In adult rhesus monkeys, we correlated seasonal changes in immune function with circannual fluctuations in immunoactive hormones. RESULTS: In neonatal marmosets, Ant reduced the number of B cells and T cells in the thymic medulla and T cells in the periarterial lymphatic sheaths (PALS) of the spleen. Ant also altered the development of, but did not permanently impair, the proliferative index (PI) of blood lymphocytes to mitogens. In vitro treatment of control lymphocytes with GnRH analogues altered their response to these proliferative agents. In neonatal rhesus monkeys, Ant treatment increased the frequency of clinical problems, lowered circulating levels of lymphocytes, total T cells, CD8+ T cells and B cells, and altered the PI of lymphocytes to mitogens. As adults, the cell- and humorally-mediated immune responses remained impaired. We also documented seasonal fluctuations in the prevalence of diseases, circulating immune cells and immune function in rhesus monkeys. The number of cases of campylobacteriosis and shigellosis was lowest in the winter and highest in the spring. Circulating numbers of white blood cells (WBC) and neutrophils and the PI of lymphocytes to mitogens were higher in the winter than in the summer. Natural killer cell activity also varied with season. Cortisol and leptin secretion exhibited circannual rhythms, rising in concert with decreasing photoperiod and increasing testicular activity in the fall. Conversely, prolactin levels declined with decreasing photoperiod and then rose in the spring. CONCLUSION: Neonatal exposure of male primates to Ant appears to alter early postnatal programming of immune function. In the rhesus monkey, immune function shows seasonal fluctuations that may be driven by circannual changes in the secretion of immunoactive hormones.

Seasonal variations in cytokine expression and cell-mediated immunity in male rhesus monkeys.

Our objectives in this study were to examine seasonal changes in immune responses including cytokine profiles of male rhesus monkeys housed under natural lighting conditions. We also monitored circannual changes in the secretion of several immunomodulatory hormones as potential mediators of the seasonal shifts in immune status. Retrospectively, the medical records of a large group of rhesus monkeys were examined to determine whether a common disease (campylobacteriosis) in this species shows a seasonal pattern of prevalence. Results of the study showed that there was a seasonal shift in the frequency of cells expressing TH1 cytokines (interleukin-2 and interferon-gamma) versus the TH2 prototype cytokine (interleukin-4) by peripheral blood mononuclear cells (PBMC) collected during the winter and summer. The frequency of TH1-type cytokine synthesis in the summer was markedly greater than in the winter whereas TH2-type cytokine expression did not vary between the two seasons. The proliferative response of PBMC to mitogens and natural killer cell activity of PBMC also varied with the season. Several hormones (testosterone, leptin, and prolactin) that modulate immune function exhibited circannual patterns of secretion. The prevalence of Campylobacter infections was higher in the spring than during the summer, fall, or winter. The data suggest that seasonal fluctuations in immune system status may alter the ability of primates to successfully respond to pathogens, and this may be related to circannual patterns of secretion of immunomodulatory hormones.

A longitudinal study of leptin during development in the male rhesus monkey: the effect of body composition and season on circulating leptin levels.

The objective of this study was to examine longitudinal changes in serum leptin concentrations during development and to correlate those changes with sexual development in male rhesus monkeys housed under natural environmental conditions. Blood samples were drawn from 8 control animals approximately every other month from 10 to 30 mo of age and thereafter monthly through 80 mo of age. Leptin levels declined through the juvenile period until the onset of puberty and were negatively correlated with body weight. Seven of the eight animals became sexually mature during the breeding season of their fourth year of life. Puberty was delayed in the other animal until the subsequent breeding season. There were no significant fluctuations in leptin levels prior to or in association with the pubertal rise in LH and testosterone (T) secretion. During the peripubertal period, levels of leptin varied between 2 and 3 ng/ml. The animal that exhibited delayed puberty had the lowest body weight and highest leptin levels during this period. With the achievement of sexual maturity, leptin levels varied seasonally, with peak levels in the late winter (Jan-Mar) and a nadir in the late summer (Aug-Sept). A late winter rise in leptin was also evident in most of the animals during Years 2 and 3, but not during Year 4. In the fall of Years 5 and 6, the seasonal rise in leptin concentrations lagged 3-4 mo behind the seasonal increase in LH and T. In the fall of Year 5, but not thereafter, leptin levels were positively related to percent body fat and negatively correlated with lean body mass. The data do not support the hypothesis that increasing leptin concentrations trigger the onset of puberty in the male rhesus monkey. During the juvenile period and after sexual maturation, but not during the peripubertal period, leptin secretion varied with season in the animals; but the environmental factors that cue or drive this rhythm remain to be determined.

Effect of neonatal treatment with a GnRH antagonist on development of the cell-mediated immune response in marmosets.

PROBLEM: We examined the effect of neonatal treatment with a gonadotropin-releasing hormone (GnRH) antagonist (antide) on the development of cell-mediated immunity in male marmosets. METHOD OF STUDY: Neonatal marmoset twins were treated with either vehicle or antide, and the proliferative response (PR) of lymphoid tissue to mitogens was assessed during infancy, the peripubertal period, and adulthood. RESULTS: Basal proliferation of peripheral blood mononuclear cells (PBMC) from treated peripubertal twins was elevated above control values, but the PR of the cells to T and B cell mitogens was subnormal. Conversely, PBMC from treated infants exhibited an enhanced PR to some of the mitogens employed. In vitro culturing of thymocytes (control or treated) from the three developmental stages with either antide or a GnRH agonist increased basal proliferation, but decreased the PR to mitogens by 60-80%. CONCLUSION: Neonatal treatment with antide alters development of, but does not permanently impair, cell-mediated immunity in the marmoset. GnRH appears to modulate immune responses throughout development in the primate.

Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress.

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-D-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was associated with a greater than 7-fold increase in serum corticosterone, and a marked reduction in serum testosterone (T) concentrations. Stress did not significantly alter hypothalamic or pituitary GluR1 and NMDA receptor mRNA levels. Although transcript levels for P450SCC and P45017alpha mRNA in the testis were unchanged in stressed rats, western blotting of testicular fractions revealed reduced amounts of P450SCC and 3beta-HSD, but not P45017alpha. The data suggest that immobilization stress reduces T production by suppressing the translation of transcripts for P450SCC and 3beta-HSD, but the attenuated LH response of stressed animals to NMDA is not mediated by altered hypothalamic or pituitary expression of GluR1 and NMDA receptor levels.

Effect of neonatal treatment with a gonadotropin releasing hormone antagonist on developmental changes in circulating lymphocyte subsets: a longitudinal study in male rhesus monkeys.

We have examined changes in circulating lymphocyte subsets from the neonatal period until adulthood (4 months until 5.5 years of age) in male rhesus monkeys, and the impact of neonatal treatment with a GnRH antagonist (Ant) or Ant and androgen (Ant/And) on these parameters. Absolute numbers of lymphocytes, B cells, total T lymphocytes, and CD4+ T cells decreased, neutrophils increased, and CD8+ T cells did not change with age. WBC counts increased between 4 mo and 2 years of age and then fell to neonatal levels over the next two years. The decline of CD4 + T cells in association with stable CD8+ T cell levels resulted in an age-related decrease in the CD4+/CD8+ T cell ratio. At 4 months of age, WBC's, lymphocytes, total T cells, CD8+ T cells and B cells were lower in Ant- and Ant/And-treated animals compared to controls. With the exception of WBC counts, these values had normalized by 2 years of age. Reduced WBC levels in treated animals persisted through adulthood. CD4+ T cell levels tended to be lower in Ant-treated and higher in Ant/And-treated animals than in controls at 4 months of age. CD4+ T cells remained lower in Ant- than in Ant/And-treated animals at most ages. The higher CD4 + T cell counts in Ant/And-treated animals resulted in an elevated CD4 + /CD8 + T cell ratio that persisted until the onset of year 5. During years 5 and 6, seasonal fluctuations in WBC's and neutrophils were observed with counts being higher in the breeding (fall) than in the nonbreeding (summer) season. The data document that developmental changes in circulating immune cells in the rhesus monkey are qualitatively similar to those reported in humans, and provide further evidence that neonatal treatment of male rhesus monkeys with Ant or Ant/And may alter early programming of the immune system.

Sexual maturation in male rhesus monkeys: importance of neonatal testosterone exposure and social rank.

In a 5-year longitudinal study, we examined the effect of disrupting the neonatal activity of the pituitary-testicular axis on the sexual development of male rhesus monkeys. Animals in a social group under natural lighting conditions were treated with a GnRH antagonist (antide), antide and androgen, or both vehicles, from birth until 4 months of age. In antide-treated neonates, serum LH and testosterone were near or below the limits of detection throughout the neonatal period. Antide + androgen-treated neonates had subnormal serum LH, but above normal testosterone concentrations during the treatment period. From 6 to 36 months of age, serum LH and testosterone were near or below the limits of detection. Ten of 12 control animals reached puberty during the breeding season of their 4th year, compared with five of 10 antide- and three of eight antide + androgen-treated animals. Although matriline rank was balanced across treatment groups at birth, a disruption within the social group during year 2 resulted in a marginally lower social ranking of the two treated groups compared with the controls. More high (78%) than low (22%) ranking animals reached puberty during year 4. During the breeding season of that year, serum LH, testosterone and testicular volume were positively correlated with social rank. Thus the lower social rank of treated animals may have contributed to the subnormal numbers of these animals reaching puberty during year 4. However, of those animals achieving puberty during year 4, the pattern of peripubertal changes in serum testosterone and testicular volume differed between control and antide-treated animals. The results appear to suggest that the disruption of normal activity of the neonatal pituitary--testicular axis retarded sexual development, but that social rank is a key regulatory factor in setting the timing of sexual maturation in male rhesus monkeys. The effect of neonatal treatment with antide and low social rank on sexual development could not be reversed by neonatal exposure to greater than normal concentrations of androgen.

Changes in lymphoid tissue after treatment with a gonadotropin releasing hormone antagonist in the neonatal marmoset (Callithrix jacchus).

PROBLEM: The effect of neonatal treatment with a gonadotropin releasing hormone (GnRH) antagonist on the morphology and distribution of lymphocytes in lymphoid tissue of the infant marmoset was examined. METHOD OF STUDY: From a screened panel of antihuman antibodies for specific immune cells, antibodies for the CD20 and CD3 antigens showed excellent reactivity with marmoset tissue. Five sets of marmoset twins were treated with either the GnRH antagonist or a vehicle from birth, and were euthanized at 7 to 9 (3 sets) or 16 to 20 weeks (2 sets) of age. The spleen, thymus, and inguinal lymph nodes from each animal were processed for immunocytochemistry, and the number of cells expressing the CD20 and CD3 antigens were quantified. RESULTS: Control twins exhibited high plasma levels of testosterone, characteristic of the neonatal period, whereas testosterone concentrations were reduced (P = 0.001) to detection limits in the GnRH antagonist-treated twins. Microscopic evaluation suggested that treatment reduced the volume and cellularity of the thymic cortex, resulting in a decrease in the cortical-to-medullary ratio. Treatment reduced (P = 0.046) the number of thymocytes expressing the B-cell antigen (CD20) and marginally lowered (P = 0.067) the number expressing the T-cell antigen (CD3) in the thymic medulla. In the spleens of treated animals, periarterial lymphatic sheaths were less prominent on microscopic examination, and there were marginally fewer (P = 0.064) CD3+ cells. Numbers of CD20+ lymphocytes in the peripheral white pulp of the spleen and in the germinal centers of the lymph nodes, or CD3+ cells in the paracortex and germinal centers of the lymph nodes, were not altered by treatment. CONCLUSION: Neonatal treatment with a GnRH antagonist may alter maturational processes for B and T cells in the thymus and spleen of the marmoset and may deprive the immune system of its normal sensitivity to GnRH at a potentially critical time in development.

Inhibin-B in the male rhesus monkey: impact of neonatal gonadotropin-releasing hormone antagonist treatment and sexual development.

We examined the effect of reversibly suppressing pituitary-testicular function during the neonatal period on developmental changes in inhibin-B and FSH secretion in male rhesus monkeys. Infants were treated with either vehicle, a GnRH antagonist (Ant) or the Ant and androgen (Ant/And) for the first 4 postnatal months, and the effects on serum inhibin-B and FSH were monitored during the neonatal and peripubertal periods. In neonates, Ant or Ant/And treatment lowered both serum FSH and inhibin-B levels. By 12 months of age, inhibin-B concentrations no longer differed across treatment groups. A major increase in inhibin-B occurred between 27-36 months of age (late prepubertal period) in all groups, but levels were lower at 33 and 36 months of age in Ant/And-treated animals than in controls. These differences most likely were related to fewer Ant/And-treated animals achieving sexual maturity during their fourth year of life. Regardless of treatment, inhibin-B levels were higher in those that were destined to become mature (in year 4) than in those that were not. During the late prepubertal period, serum inhibin-B was positively correlated with age and testicular volume, but not with serum LH or testosterone. After this period (39-52 months of age), inhibin-B no longer correlated with these parameters. FSH levels were near or below detection limits in most peripubertal animals, but FSH was detectable in fewer samples from control than treated animals. The data suggest that inhibin-B secretion in the neonate is driven by gonadotropin secretion, but during the juvenile hiatus in gonadotropin secretion, the monkey testis continues to produce substantial amounts of this hormone.

Embryonic survival in the uterus of ewes inseminated at the uterotubal junction on Day 32 post partum.

Experiments were conducted to determine 1) if pregnancy initiated on Day 32 post partum would be maintained until lambing, 2) if there is a difference in the ability of the previously gravid or nongravid uterine horn to maintain pregnancy, and 3) if season has an effect on embryo loss. Estrus was induced in ewes on Day 32 post partum. At estrus, ewes were inseminated surgically at the uterotubal junction and assigned to the following groups: 1) inseminated at estrus and laparotomized on Day 3 to collect embryos for determination of fertilization rate (C), 2) inseminated in the previously gravid uterine horn (PG), 3) inseminated in the previously nongravid uterine horn (NG), and 4) inseminated when both horns were previously gravid (BG). Ewes pregnant in the PG, NG and BG groups were allowed to lamb. Conception rate in Group C at embryo collection was 70%. Embryo loss, based on concentrations of progesterone at Day 18 post insemination, was 43, 19 and 18% in the BG, NG and PG group, respectively. The high embryo loss in Group BG occurred only during the breeding season. Only 24% of the ewes that had been inseminated lambed. This was due to the prepartum loss of embryos and fetuses (47, 48 and 33% in Group BG, NG and PG, respectively. In conclusion, the detrimental effects of the uterus on embryo survival was evident within 18 d post insemination in Group BG (breeding season), and embryo loss prior to lambing was high in all the treatment groups (both seasons).

Neonatal testosterone and handedness in yearling rhesus monkeys (Macaca mulatta).

This study investigated the relationship between neonatal testosterone (T) and hand bias in young rhesus monkeys (Macaca mulatta). Subjects (n = 8 per group) included: neonatally androgen-suppressed males, using a Nal-Lys gonadotropin releasing hormone (GnRH) antagonist (Antide); androgen-suppressed males receiving T replacement by a long-acting T preparation (CDB); control males; and control females. Antide suppressed T to the female range, whereas CDB replacement produced supranormal levels. Visually guided reaching, in a social context, showed a population-level left-hand bias. Males with elevated T did not show a stronger left-hand bias than males with normal T, but did show a stronger bias for the preferred hand whether left or right. Males with Antide-suppressed T showed an intermediate degree of hand bias. Results suggest that high neonatal T levels affect laterality and raise the possibility that GnRH analogues influence brain development. These data suggest a broad influence of the CNS-pituitary-testicular axis on brain asymmetries and provide support for an early neonatal period of T-influenced brain differentiation.

Acute immobilization stress alters LH and ACTH release in response to administration of N-methyl-D,L-aspartic acid in peripubertal and adult male rats.

We have evaluated the effects of stress on the pituitary hormone response to N-methyl-D,L aspartic acid (NMA) in peripubertal (43 day old) and adult (80 day old) male rats. Animals were stressed by immobilization for 3 h. Fifteen min prior to the end of the stress period, animals were injected sc with either vehicle or increasing doses of NMA (10, 30 or 60 mg/kg BW). Animals were sacrificed 15 minutes later. A significant rise in plasma LH in response to NMA occurred at a lower dose (10 versus 60 mg/kg BW), and the magnitude of the response was greater overall in peripubertal than in adult unstressed rats. Stress altered the LH response to NMA in both age groups. In peripubertal stressed rats, NMA did not induce a rise in plasma LH levels. In adult rats exposed to stress, the magnitude of the LH response to 60 mg/kg BW of NMA was lower than in unstressed animals. NMA administration triggered a rise in plasma ACTH levels in unstressed rats of both age groups, but the magnitude of the rise was greater in adults than in peripubertal animals. Stress reduced the ACTH response to NMA administration in both age groups. Plasma FSH concentrations were higher in peripubertal animals than in adults. NMA and stress were without major effect on plasma FSH levels in either age group. These data suggest that excitatory amino acids (EAA) form an important component in the regulatory processes governing the release of LH and ACTH, and that the maturational stage of the central nervous system and exposure to stress alter the LH and ACTH response to EAA.

Effect of a peripheral and a central acting opioid antagonist on the testicular response to stress in rats.

The possible involvement of opioid receptors in mediating the inhibitory effects of immobilization stress on testicular steroidogenesis was determined in adult male rats. Unstressed controls and animals exposed to 3 h of immobilization stress were injected subcutaneously with either vehicle or 1 or 10 mg/kg body weight (BW) of naloxone or naltrexone methobromide (NMB; an opioid receptor antagonist that does not cross the blood-brain barrier) at the beginning of and at 1.5 h of the stress period. Animals were sacrificed at 2 h (30 min after the second injection of antagonist) or 3 h (90 min after the second injection of antagonist) of stress. Plasma LH was not affected by stress, but 30 min after naloxone (1 or 10 mg/kg BW) injection, LH was elevated in both control and stressed rats above levels in vehicle-injected animals. By 90 min after naloxone injection, plasma LH had declined to levels comparable to those in vehicle-injected animals. NMB had no effect on plasma LH concentrations in either control or stressed rats. Three hours of stress reduced plasma testosterone (T) levels by 60% in vehicle-injected animals. This effect of stress on plasma T levels was antagonized by the 10 mg/kg BW dose of naloxone and 1 or 10 mg/kg BW of NMB. The ability of naloxone to reverse the effect of stress on plasma T levels was likely related to its ability to stimulate LH secretion, but NMB normalized plasma T values in stressed animals without altering plasma LH concentrations. Only the highest dose of NMB increased plasma T levels in unstressed control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal treatment with luteinizing hormone-releasing hormone analogs alters peripheral lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys.

We examined the effect of treatment with a LH-releasing hormone (LHRH) agonist (Ag), antagonist (Ant), or Ant and androgen (Ant/And) for the first 4 months of postnatal life on lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys. We also determined the effect of 9 weeks of Ant treatment on lymphocyte subsets in adult male monkeys. Adult male monkeys that had been treated neonatally with an Ag had increased levels of CD8-positive (CD8+) T-cells and reduced levels of B-cells compared to vehicle-treated controls. Lymphocytes from these animals also showed an elevated proliferative response to a variety of mitogens compared to cells from control animals. Antibody production in response to tetanus toxoid was normal in treated animals. Other neonates treated with Ant/And exhibited subnormal levels of lymphocytes, CD8+ T-cells, and B-cells at 4 months of age. Similar changes, but of lesser magnitude, were observed in animals treated with Ant alone. At 6 months of age, lymphocytes from both groups of Ant-treated monkeys exhibited an above normal proliferative response to streptolysin-O, but not to other mitogens. At 18 months of age, animals treated with Ant alone produced more antitetanus antibody in response to a tetanus toxoid booster than the controls or Ant/And-treated animals. Ant treatment was without major effect on lymphocyte subsets in adult monkeys. Serum LH and testosterone levels declined, and there was a small but significant increase in B-cells, lymphocytes expressing the interleukin-2 receptor, and the CD4+/CD8+ T-cell ratio during treatment, but these parameters normalized during the posttreatment period. The data suggest that chronic neonatal treatment with an Ag or Ant alters the development of immune system responses in male primates. The significance of these changes and their impact on the ability of these animals to respond to pathogenic agents is under investigation.

Neonatal treatment of male monkeys with a gonadotropin-releasing hormone agonist alters differentiation of central nervous system centers that regulate sexual and skeletal development.

Male rhesus monkeys treated continuously with a GnRH agonist for the first 4 months of postnatal life exhibited a delay in the onset of puberty and an attenuated peripubertal rise in testosterone (T) secretion. The objectives of the current study were to determine whether these effects on sexual development were permanent and whether the hypothalamic-pituitary-testicular axis was functioning normally in these animals as adults. Neonatal GnRH agonist treatment delays but does not permanently block sexual maturation in male monkeys. Treated animals that did not show a pubertal rise in serum T during the breeding season of their 4th year exhibited a seasonal but subnormal elevation of serum T during the subsequent breeding season. Growth of the skeleton was diminished as evidenced by shorter adult crown-rump, tibia, and femur length and reduced bone mineral density of the humerus and lumbar spine. The magnitude of the serum LH and T response to iv pulses of GnRH [50 ng/kg body weight (BW)] and naloxone (1 mg/kg BW) did not differ between control and treated animals during the nonbreeding or breeding season at 6 yr of age. Conversely, treated animals showed a subnormal serum LH and T response to N-methyl-D,L-aspartic acid (5 mg/kg BW iv) during the nonbreeding season. These data suggest that adult monkeys treated neonatally with a GnRH agonist exhibit subnormal sensitivity of the central nervous system to one or more excitatory amino acids (e.g. aspartate or glutamate). Thus, abolishing neonatal activation of the pituitary-testicular axis with a GnRH agonist may permanently alter differentiation of central nervous system centers that are either involved in GnRH secretion or govern this process.

Preservation of bone mass in hypogonadal female monkeys with recombinant human growth hormone administration.

This study examined the effect of human recombinant GH supplementation on bone loss in female monkeys made hypogonadal with GnRH agonist (GnRH-Ag). Animals were randomly assigned to three treatment groups: vehicle, GnRH-Ag, and GnRH-Ag and GH. After an initial 5-month pretreatment period during which all animals were maintained on a normal monkey chow diet containing a high level of calcium (1%) animals were maintained on a normal monkey chow diet containing a high level of calcium (1%), animals were shifted to a lower calcium diet (0.1%) for 4 to 5 months before the beginning of treatment and were maintained on this diet throughout the remainder of the study. Monkeys were treated continuously for 10 months with 25 micrograms/day GnRH-Ag or vehicle. GH was administered by im injection three times per week at a dose of 100 micrograms/kg body wt/day. Animals treated with GnRH-Ag were amenorrheic throughout the treatment period, and serum estradiol and progesterone levels were below minimum levels of detection. Vehicle-treated animals continued to cycle throughout the study. Monkeys treated with GnRH-Ag alone showed a significant decline (12%) in bone mineral density (BMD) of the lumbar spine. BMD was reduced below pretreatment levels from 6 months of GnRH-Ag treatment through 3 months post treatment in this group. GH supplementation reduced the decline of BMD in GnRH-Ag-treated monkeys. BMD did not change significantly with time in the GH-supplemented group. BMD values in GH supplemented animals between 5 and 10 months of the treatment period exceeded levels in animals treated with GnRH-Ag alone, but BMD levels during this interval were lower than in the vehicle-treated group. In the vehicle-treated group, there was small, but significant, increase in BMD over the course of the study. Serum osteocalcin concentrations were elevated above pretreatment values after 6 and 9 months of GnRH-Ag treatment alone or with GH supplementation, but did not change in vehicle-treated animals. GH also increased serum insulin-like growth factor 1 levels. In response to the lower calcium diet, serum PTH levels increased approximately 200% in vehicle-treated monkeys and animals treated with GnRH-Ag alone. GH attenuated this increase in serum PTH. The data indicate that the level of calcium in the diet of adult monkeys can be reduced more than 10-fold without affecting lumbar BMD provided ovarian function is normal, but if animals are made hypogonadal with a GnRH-Ag, bone mass declines.(ABSTRACT TRUNCATED AT 400 WORDS)

Cervical dilation, conception rate, and concentrations of progesterone and estradiol-17B in postpartum ewes treated with porcine relaxin.

Experiments were conducted to determine the effects of porcine relaxin (pRLX) on cervical dilation and conception rates in postpartum ewes. In Experiment 1, ewes received medroxyprogesterone acetate (MAP) sponge on day 16 (day 0 = lambing) and 750 IU pregnant mare serum gonadotropin (PMSG) at sponge removal on day 30. Control ewes received saline and relaxin-treated (RLX) ewes received 0.5 mg pRLX (>/= 3000 U/mg) i.m. at 24 h and 1.0 mg pRLX at 36 h after PMSG. All ewes were inseminated (Al) at 55 h after PMSG with 0.4 ml fresh semen. The proportion of RLX treated ewes (6 6 ) in which the cervix was penetrated was greater (P < 0.05) than in Control ewes (0 5 ). However, ova recovery rate was lower (P < 0.05) for RLX ewes (1 6 ) than for control ewes (5 5 ). In Experiment 2, ewes between Days 90 to 120 post partum received MAP sponges for a period of 8 d and 750 IU PMSG at sponge removal. Control ewes (n = 9) received saline; RLX-1 ewes (n = 8) received 0.5 mg pRLX at 24 h and an additional 0.5 mg pRLX at 36 h after PMSG; and RLX-1.5 ewes (n = 9) received 0.5 mg pRLX at 24 h and an additional 1.0 mg pRLX at 36 h after PMSG. Ewes were mated to rams at estrus, and cervical dilation was checked at 55 h after PMSG. The cervix could not be penetrated in any of the ewes. Conception rates on Day 26 were 66, 56 and 63% for control, RLX-1 and RLX-1.5 groups, respectively. These results demonstrate that the effect of relaxin on cervical dilation and conception rate is dependent upon the postpartum stage of the ewes.

Conception rates in early postpartum ewes bred naturally or by intrauterine insemination.

Ewes were treated with a medroxyprogesterone acetate (MAP) sponge for 8 d followed, at sponge removal, with 500 IU pregnant mare serum gonadotropin (PMSG) at d 30, 40 or 50 (d 0 = lambing) to induce estrus. Dry and lactating ewes were divided into equal numbers at each postpartum day and bred at estrus. Conception rates and number of accessory sperm were determined by flushing the oviducts 3 d after mating and examining the recovered ova. There was no effect (P greater than .05) of lactational status on conception rates. Conception rates increased (P less than .05) from d 30 (10%) to d 40 (45%) and from d 40 to d 50 (80%). There were fewer (P less than .05) ova with accessory sperm (5/26) in d-30 ewes compared with d-40 (10/27) or d-50 (12/24) ewes. In Exp. 2, ewes were assigned to two groups after receiving PMSG on d 30: 1) mated naturally or 2) inseminated during laparotomy near the uterotubal junction (UTJ). Dry and lactating ewes were divided evenly within each of the two treatments. Oviducts were flushed and ova were examined for cleavage. The conception rate was 60% in ewes that were inseminated in the UTJ vs 10% in ewes mated to rams (P less than .05). Lactational status had no effect on results. In conclusion, conception rates in postpartum ewes treated with MAP sponge and PMSG increased from postpartum d 30 to d 50 with natural breeding, and d-30 conception rates were increased over natural mating by insemination into the uterine horn near the UTJ.