Effects of cocaine on basal and pulsatile prolactin levels in rhesus monkeys.

OBJECTIVES: Cocaine abuse is often associated with reproductive cycle dysfunction including altered menstrual cyclicity and decreased ovulation rates. Cocaine might also alter prolactin (PRL) secretion, presumably through the effects of this drug on hypothalamic dopamine, the primary factor regulating pituitary PRL secretion. We assessed basal and pulsatile PRL levels to determine whether hyperprolactinemia is associated with cocaine-induced disruption of menstrual cyclicity in rhesus monkeys. METHODS: Normally cycling, drug-naïve monkeys were studied. Cocaine-treated animals were pair-fed with controls to minimize cocaine-related differences in caloric intake. Twenty-eight monkeys were randomized to receive daily intravenous (iv) infusion of saline or cocaine (1, 2, or 4 mg/kg) on cycle days 2-14. Daily blood samples were obtained through indwelling catheters for measurement of ovarian steroids, gonadotropins, and PRL. Laparoscopy was performed 2 days after the midcycle estradiol surge to document ovulation. Sixteen other monkeys were randomized to receive daily iv infusion of saline or cocaine (4 mg/kg). Blood samples were obtained every 15 minutes for 8 hours in the early (cycle day 1-5), mid- (cycle day 6-10), and late (cycle day 11-15) follicular phase. Plasma was assayed for PRL, and pulses were identified by cluster analysis. RESULTS: All seven control monkeys had laparoscopically confirmed ovulation compared to two of seven monkeys receiving 1 mg/kg, three of seven monkeys receiving 2 mg/kg, and one of seven receiving 4 mg/kg of cocaine hydrochloride. Cycle length was normal in six of seven controls, and in one of seven, two of seven, and two of seven monkeys receiving the 1, 2, and 4 mg/kg of cocaine, respectively. Estradiol levels were significantly higher in controls versus cocaine-treated monkeys, but there was no difference in basal gonadotropin levels during treatment. Mean PRL levels during treatment were significantly lower (P <.05) in controls (4.6 +/- 0.2 ng/mL) as compared to monkeys receiving 1 (6.5 +/- 0.6 ng/mL), 2 (6.1 +/- 0.4 ng/mL), and 4 mg/kg (7.2 +/- 0.6 ng/mL) of cocaine. There was no significant difference in PRL pulse amplitude or frequency between controls and cocaine-treated monkeys during each cycle phase. CONCLUSIONS: Circulating PRL levels were slightly higher in monkeys receiving cocaine during the follicular phase. Although this increase was statistically significant, PRL levels remained well within the euprolactinemic range in cocaine-treated monkeys.

Cocaine impairs ovarian response to exogenous gonadotropins in nonhuman primates.

OBJECTIVE: Determine whether cocaine directly impairs ovarian steroid production and ovulation. METHODS: Normally cycling adult female rhesus monkeys received daily intravenous normal saline (control; n = 8) or cocaine (4 mg/kg; n = 8) through the follicular phase. Monkeys were injected daily with human menopausal gonadotropin (hMG; Pergonal) at a dose of 6 IU/kg intramuscularly beginning on cycle day 2. Daily blood samples were obtained, and serum estradiol (E(2)) and progesterone (P(4)) were measured by radioimmunoassay. When serum levels of E(2) declined, plateaued, or exceeded 600 pg/mL, laparoscopy was performed to count the number of follicles. If no new corpus luteum was present, monkeys were injected intramuscularly with 1000 IU of hCG. Laparoscopy was repeated 2 days later to document the number of ovulatory stigma. RESULTS: During ovarian stimulation, cocaine-treated monkeys required an average additional 1.5 days of hMG injections (P =.01), and this resulted in a greater total dose of hMG compared with control monkeys (351 +/- 16 IU versus 297 +/- 15 IU [mean +/- standard error of the mean], P =.03). For spontaneous and hCG-triggered ovulation, the number of ovulatory stigma was significantly lower (P <.003) in the cocaine-treated versus control monkeys (16 versus 31). Peak E(2) levels were significantly (P =.05) lower in cocaine-treated monkeys compared with controls. Luteal phase P(4) levels were lower in the cocaine-treated monkeys, but the difference was not statistically significant when compared with controls. CONCLUSION: Cocaine impaired ovarian responsiveness to exogenous gonadotropins and decreased ovulatory stigma in nonhuman primates. These findings suggest that cocaine has direct ovarian effects.

Impact of implementation of an embryo storage fee on embryo disposal activity.

OBJECTIVE: To examine the impact of implementation of a new fee for continued storage of cryopreserved embryos on the rate of requests for disposal of embryos. DESIGN: Retrospective cohort study. SETTING: A university-based assisted reproduction program. PATIENT(S): All patients with cryopreserved embryos. INTERVENTION(S): Implementation of a semiannual embryo storage fee of $100 to cover administrative and laboratory costs. MAIN OUTCOME MEASURE(S): The number of embryo disposal requests before and after implementation of the embryo storage fee was compared in relation to the activity of the cryopreserved embryo program as measured by number of frozen embryo transfers. RESULT(S): Annual requests for embryo disposal from 1992 through 1997 ranged from zero to three, which represented 0-5% of the annual frozen embryo program activity. In contrast, a significantly higher number of disposal requests (10, representing 18% of program activity) were received in 1998. CONCLUSION(S): Fees for storage of cryopreserved embryos seem to influence patients' decisions about disposal of cryopreserved embryos.

Medical care program compliance--a year 2000 recipient perspective of Medicare claims.

This article provides a brief assessment of patient and provider views and concerns regarding reimbursements under the Medicare program. Specifically targeted is the payment of pharmaceutical claims. Also addressed are the ongoing and respective responsibilities of individual clinical providers, associated hospitals, and recipients of care. A summation of significant results of direct interviews and follow-up discussions with 10 Medicare recipients also is provided.

Survival of cryopreservation and thawing with all blastomeres intact identifies multicell embryos with superior frozen embryo transfer outcome.

OBJECTIVE: To assess the impact of survival of cryopreservation and thawing with all blastomeres intact on the outcome of multicell frozen ET. DESIGN: Retrospective study. SETTING: Academic assisted reproductive technology program. PATIENT(S): One hundred sixteen exclusively multicell frozen ETs in 78 patients. INTERVENTION(S): Frozen ET. MAIN OUTCOME MEASURE(S): Relation of embryonic blastomere survival to the outcome of frozen ET (i.e., pregnancy). RESULT(S): When at least one embryo survived with all blastomeres intact, the total pregnancy rate (biochemical, clinical, or delivered) was 37.7%, the clinical pregnancy rate was 24.6%, and the delivered pregnancy rate was 18.8%. When no embryo survived with all blastomeres intact, the corresponding rates were 10.6%, 8.5%, and 6.4%. The differences in the total pregnancy rate and the clinical pregnancy rate were statistically significant. The delivered pregnancy rates approached statistical significance. CONCLUSION(S): Multicell embryonic survival of cryopreservation and thawing with all blastomeres intact identifies embryos with superior developmental potential.

Cytogenetic and fertility studies of a rheboon, rhesus macaque (Macaca mulatta) x baboon (Papio hamadryas) cross: further support for a single karyotype nomenclature.

Historically, two different numbering systems have been used to describe the baboon and macaque karyotypes. However, G-banding studies and, more recently, fluorescence in situ hybridization results have shown that the two karyotypes are virtually identical. To confirm this hypothesis, cytogenetic analysis of an unusual animal, a rheboon, was undertaken. The rheboon reported here, an 18-year-old male, is the only long-term survivor of 26 pregnancies resulting from matings between female baboons (Papio hamadryas) and male rhesus macaques (Macaca mulatta). A G-banded karyotype was prepared from the rheboon and compared with the karyotypes of the two parental species. Spectral karyotyping (SKY) was carried out on the rheboon chromosomes, and the results were compared with SKY studies reported for the baboon and with CISS (chromosome in situ suppression) studies in the rhesus macaque. No differences were detected in any of the rheboon's pairs of autosomes, reinforcing the apparent identity of the two parental karyotypes. Based on these results, we argue that a single karyotyping system should be adopted for the two species. Fertility studies were initiated to determine if the rheboon is sterile, as are most hybrid animals. Two semen ejaculates were devoid of sperm. A testicular biopsy revealed hypoplasia of the seminiferous tubules with few Leydig cells and large lumena. Meiotic arrest occurred during meiosis I, resulting in absence of mature spermatozoa. Thus, the testicular and meiotic findings in the rheboon were similar to those observed in other hybrids, even though the parental karyotypes appear identical.

In vitro maturation of baboon oocytes retrieved at the time of cesarean section.

OBJECTIVE: To investigate the feasibility of oocyte retrieval at the time of cesarean delivery and the potential of such oocytes to undergo nuclear maturation in vitro using a baboon model and an established culture system. DESIGN: Randomized, controlled animal study. SETTING: Research foundation and university research laboratory. ANIMAL(S): Mature pregnant baboons. INTERVENTION(S): In vitro culture of aspirated oocytes with or without epidermal growth factor (EGF). MAIN OUTCOME MEASURE(S): Oocyte yield, germinal vesicle breakdown, polar body extrusion. RESULT(S): A total of 246 oocytes were retrieved (mean, 35; range, 14-67). Eighty-seven oocytes (35%) underwent germinal vesicle breakdown and 72 oocytes (29%) extruded a polar body. A chi2 analysis revealed no significant effect of EGF on outcome parameters. No effect of gestational age or maternal age on oocyte yield or development was observed. CONCLUSION(S): A sizeable proportion of oocytes obtained from puerperal primates exhibited the capacity to undergo nuclear maturation in vitro.

Multiple elements influence transcriptional regulation from the human testis-specific PGK2 promoter in transgenic mice.

The PGK2 gene is expressed in a strictly tissue-specific manner in meiotic spermatocytes and postmeiotic spermatids during spermatogenesis in eutherian mammals. Previous results indicate that this is regulated at the transcriptional level by core promoter sequences that bind ubiquitous transcription factors and by sequences in a 40-base pair (bp) upstream enhancer region (E1/E4) that bind tissue-specific transcription factors. Transgenic mice carrying different PGK2 promoter sequences linked to the chloramphenicol acetyltransferase (CAT) reporter gene, one containing only the 40-bp E1/E4 enhancer sequence plus the core promoter and two containing 515 bp of PGK2 promoter but with either the E1/E4 enhancer region or the Sp1-binding site in the core promoter disrupted by in vitro mutagenesis, all showed levels of expression reduced to less than half that of the wild-type 515 PGK2/CAT transgene. These results indicate that multiple factor-binding regions normally regulate initiation of transcription from the PGK2 promoter. The single disruption of any one of these binding activities reduced, but did not abolish, transgene expression. This is consistent with an "enhanceosome"-like function in this promoter involving multiple bound activator proteins that interact in a combinatorial manner to synergistically promote testis-specific transcription.

Low-dose follicular-phase cocaine administration disrupts menstrual and ovarian cyclicity in rhesus monkeys.

OBJECTIVE: To evaluate the effects of daily low-dose follicular-phase cocaine administration on menstrual cyclicity, ovulation rates, corpus luteum function, and hormone levels in rhesus monkeys. METHOD: Normally cycling, drug-naive, adult rhesus monkeys were randomized to receive either 1 mg/kg of cocaine (n = 7), 2 mg/kg of cocaine (n = 7), or normal saline (n = 7) daily on cycle days 2 to 14. Daily blood samples were obtained through indwelling catheters for measurement of serum gonadotropins and ovarian steroids. Daily vaginal swabs were obtained to determine onset of menses. Laparoscopy was performed 2 days after the midcycle estrogen peak to document ovulation. Daily caloric intakes as well as pretreatment and posttreatment weights were recorded. RESULTS: Two of seven monkeys receiving 1 mg/kg per day and two of seven monkeys receiving 2 mg/kg per day of cocaine had timely ovulation and normal menstrual cycle lengths. One monkey receiving the 2-mg/kg dose ovulated on cycle day 24 and had a short luteal phase (7 days) with a mean progesterone level of 2.4 ng/mL. All seven saline-treated control monkeys ovulated normally; the mean cycle length was 29 days and all had adequate luteal phases. The difference in ovulation rates between cocaine-treated and control monkeys was statistically significant (P = .003). There were no differences in basal levels of LH or FSH between treatment groups. There were no significant differences in weight change or caloric intake among groups. One third of the subsequent menstrual cycles in cocaine-treated monkeys were of abnormal duration. CONCLUSION: Daily low-dose follicular-phase cocaine administration disrupts menstrual cyclicity and folliculogenesis. This effect is independent of weight loss, caloric intake, and basal gonadotropin levels. Cocaine exposure may have a persistent effect on menstrual and ovarian cyclicity in some monkeys.

Cocaine impairs follicular phase pulsatile gonadotropin secretion in rhesus monkeys.

OBJECTIVE: To assess cocaine's effect on follicular phase pulsatile gonadotropin secretion in normally cycling rhesus monkeys. METHODS: Sixteen monkeys were paired by body weight and randomized to receive intravenous saline (n = 8) or cocaine (4 mg/kg, n = 8) daily on cycle days 2 to 14. Monkeys were chronically cannulated to allow frequent blood collections without anesthesia. Blood samples were obtained every 15 minutes for 8 hours in early (EFP; cycle days 1 to 5), mid-(MFP; cycle days 6 to 10), and late (LFP; cycle days 11 to 15) follicular phase. Plasma concentrations of LH, FSH, and estradiol-17 beta (E2) were determined by radioimmunoassay. Pulses were identified by cluster analysis. Statistical differences were determined by analysis of variance (ANOVA) and Sidak's multiple comparison test. RESULTS: Seven out of eight monkeys in the control group demonstrated timely ovulation. Only one monkey in the cocaine-treated group ovulated. Similar gonadotropin pulse intervals (70 to 90 minutes) were observed throughout the follicular phase in both the controls and cocaine-treated monkeys. LH and FSH pulse amplitudes increased significantly from the EFP/MFP to the LFP in controls. In cocaine-treated monkeys, gonadotropin pulse amplitudes remained at EFP/MFP levels throughout the study period. The mean gonadotropin pulse amplitude and the mean E2 levels in the LFP were significantly greater in controls as compared with cocaine-treated monkeys (P < .001). CONCLUSION: These findings demonstrate that cocaine suppresses the normal increase in LH and FSH pulse amplitude seen in the LFP. Further studies are in progress to determine the mechanism of cocaine's disruption of the hypothalamic-pituitary-ovarian axis.

Effects of follicular-phase cocaine administration on menstrual and ovarian cyclicity in rhesus monkeys.

OBJECTIVE: The purpose of this study was to evaluate the effects of daily follicular-phase cocaine administration on menstrual cyclicity, gonadotropin and ovarian steroid levels, ovulation rates, and corpus luteum function in cycling rhesus monkeys. STUDY DESIGN: Thirteen normally cycling, drug-naive adult rhesus monkeys were randomized to receive daily intravenous injections of either 4 mg/kg cocaine or an equal volume of saline solution. Treated animals were yoked to pair-fed controls to minimize differences in caloric intake. Daily blood samples were obtained through indwelling catheters for measurement of serum gonadotropin and ovarian steroid levels. Daily vaginal swabs were obtained to determine the onset of menses. Laparoscopy was performed 2 days after the midcycle estrogen peak to check for ovulation. Daily caloric intakes and pretreatment and posttreatment weights were recorded. RESULTS: All six of the control monkeys had laparoscopically confirmed ovulation compared with one of seven in the cocaine-treated group (p < 0.004). Cycle length was normal in five of six controls versus one of seven cocaine-treated monkeys. Estradiol levels were significantly higher in the controls versus the cocaine-treated monkeys (p = 0.01) during the first 14 days of the treatment cycle. There were no differences in basal plasma gonadotropin levels between groups. Luteal-phase lengths and luteal-phase plasma progesterone levels were similar in the controls and the single ovulatory cocaine-treated monkey. There were no significant differences in weight change or caloric intake between the two groups. CONCLUSIONS: Daily follicular-phase cocaine administration disrupts menstrual cyclicity and folliculogenesis independent of weight loss, caloric intake, and basal gonadotropin levels.

Effects of hirudin (thrombin specific inhibitor) in zebrafish embryos: a developmental role for thrombin.

To address the role of thrombin in early embryogenesis, hirudin, a thrombin specific inhibitor was microinjected into developing zebrafish embryos to inhibit the temporal activity of thrombin during early embryonic development. We found that the fibrin forming activity is inhibited by the presence of hirudin. We also found that hirudin affects development in zebrafish embryos suggesting thrombin's role in early embryogenesis. This ability to inhibit thrombin activity in developing embryos should facilitate studies on identifying signal transduction pathways affected by thrombin during embryogenesis.

The glycoprotease of Pasteurella haemolytica A1 eliminates binding of myeloid cells to P-selectin but not to E-selectin.

HL-60 cells and neutrophils treated with the glycoprotease from Pasteurella haemolytica A1, an enzyme which is specific for O-sialoglycoproteins, were found to be incapable of binding P-selectin but still bound E-selectin. Comparative analysis of [35-S] cysteine labeled proteins from HL-60 cells by 2-dimensional electrophoresis indicated that two major proteins with M(r) 100 and 115 kd were significantly removed from cells which had been treated.

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Rabbit endosalpinx inhibits implantation in vitro.

OBJECTIVE: To evaluate the ability of rabbit endometrial or endosalpingeal cells to support implantation in vitro and to assess the effects of endosalpinx and endometrium-conditioned media (CM) on blastocyst-endometrial cell interaction. DESIGN: In one experiment, rabbit blastocysts were co-cultured in vitro with endometrial or endosalpingeal cells growing on Matrigel-coated plastic culture plates or Millicell-HA inserts. In a second experiment, rabbit blastocysts were co-cultured with endometrial cells in the presence of fresh medium or of endosalpinx- or endometrial-CM. After 48 or 72 hours attachment to the cell monolayer was evaluated. RESULTS: Blastocysts in co-culture attached to endometrial but not to endosalpingeal monolayers. The addition of CM from cultured endosalpinx significantly decreased embryo attachment to endometrial cells in culture. CONCLUSIONS: These findings in vitro agree with the observation that rabbit endosalpinx in vivo does not support embryo implantation and support the hypothesis that rabbit endosalpinx secretes a factor that prevent tubal implantation.

Rabbit endosalpinx suppresses ectopic implantation.

This study was undertaken to elucidate the mechanism underlying the absence of oviductal ectopic pregnancies in infraprimates. Endosalpingeal circumferential grafts were substituted for endometrium in a group of rabbits. The endosalpingeal grafts interfered with implantation as evidenced by four observations: (1) The nidation indices were lowered, (2) no implantations occurred on the grafted endosalpinx, (3) unattached blastocysts were found in animals grafted with endosalpinx, and (4) the implantation sites were significantly smaller in uterine horns containing endosalpingeal grafts. These findings suggest the presence of a factor in rabbit endosalpinx that actively suppresses ectopic implantation in the oviduct.

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice.

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.

Endosurgical electrocoagulation of the mesovarium as an alternative method of inducing surgical menopause in monkeys.

This study in the primate demonstrates that electrocoagulation of the mesovarium under endoscopic visualization could successfully replace conventional surgical excision of the ovary.

A new method of transcervical female sterilization: preliminary results in rabbits.

In search of an outpatient procedure for female sterilization, the authors performed the following animal study. Two groups of seven rabbits were subjected to laparotomy, hysterotomy, and hysteroscopy. The uterotubal junction (UTJ) was destroyed with bipolar electrical current, and a plug (Aqualloy, Drachten, The Netherlands) was inserted. The plug on the right side contained quinacrine (Q) in group A and platelet extract (PE) in group B. Histologic assessment by serial sections indicated occlusion of the UTJ in all but one case (96% success rate). This one failure is attributed to the technical difficulties associated with hysteroscopy in the rabbit. The authors project that this method may have the potential of becoming an acceptable outpatient procedure for female sterilization in the human.

Effect of ovarian endometriosis on ovulation in rabbits.

To study the relationship between endometriosis and ovulatory dysfunction, we induced ovarian endometriosis in the rabbit model Adipose tissue was placed in the contralateral ovary as a control. Ovulation was induced with human chorionic gonadotropin, and ovulation points were counted before and after induction of endometriosis. Periovarian adhesions were graded, and ovaries were histologically examined. A significant decrease in the number of ovulation points was observed in ovaries with endometrial tissue (p = 0.001) but not in ovaries that contained adipose tissue (p = 0.095). Periovarian adhesions decreased the number of ovulation points (p less than 0.01) in ovaries that contained adipose or endometrial tissues. Multivariate analysis demonstrated that an increase in adhesion severity was correlated with a decrease in the number of ovulation points (p less than 0.05), but endometrial tissue was not (p = 0.45). We conclude that, in the rabbit model, minimal ovarian endometriosis impairs ovulation primarily through a mechanism related to periovarian adhesions.