In vitro progesterone production by luteinized human mural granulosa cells is modulated by activation of AMPK and cause of infertility.

BACKGROUND: Mural granulosa cells from IVF patients were provided by the West Virginia University Center for Reproductive Medicine in Morgantown, WV. The effect of adenosine monophosphate activated protein kinase (AMPK) activation, primary cause of infertility, age, BMI, and pregnancy outcome on production of progesterone were examined separately. METHODS: Isolated mural sheets from IVF patients (n = 26) were centrifuged, supernatant discarded, and the pellet re-suspended in 500 µl of DMEM/F12. Mural granulosa cells were plated at 10,000 cells/well in triplicate per treatment group with 300 µl DMEM/F12 media at 37 °C and 5% CO2 in a humidified incubator to permit luteinization. Four days after initial plating, cells were treated with either an AMPK inhibitor, DM; an AMPK activator, AICAR; or hCG. Cells were cultured for 24 h after treatment when medium was collected and frozen at -20 °C until assayed for P4 by radioimmunoassay. RESULTS: The AMPK activator, AICAR, inhibited P4 production (P < 0.001), whereas the AMPK inhibitor, DM, did not affect basal P4 (P < 0.05). Progesterone production increased when cells from patients whose primary cause of infertility was a partner having male infertility were treated with hCG compared to control (P = 0.0045), but not in patients with other primary infertility factors (P > 0.05). Additionally, hCG increased P4 production in patients between the ages 30-35 (P = 0.008) and 36-39 (P = 0.04), but not in patients ages 25-29 (P = 0.73). Patients with normal BMI had increased P4 production when treated with hCG (P < 0.0001), however there was no change in P4 production from cells of patients who were overweight or obese (P > 0.05). Cells from patients who became pregnant to IVF had greater P4 production when stimulated with hCG than those who did not become pregnant when compared to controls (P > 0.05). CONCLUSIONS: Understanding how AMPK activation is regulated in ovarian cells could lead to alternative or novel infertility treatments. Human mural granulosa cells can serve as a valuable model for understanding how AMPK affects P4 production in steroidogenic cells. Additionally, when stimulated with hCG, P4 production by mural granulosa cells differed among infertility type, age, BMI, and pregnancy outcome.

The effects of cyclic adenosine monophosphate, forskolin, and theophylline on motility parameters in gossypol-treated human sperm.

OBJECTIVE: To examine the effect of gossypol on human sperm in vitro and the mechanism for the effect. DESIGN: Fresh sperm ejaculates obtained from normal donors to the University of Kentucky Andrology Donor Program were exposed to gossypol. Motility was studied manually and using computer-assisted sperm analysis. In subsequent experiments, the effects of forskolin, theophylline, and cyclic adenosine monophosphate (cAMP) on sperm motion were measured. SETTING: University of Kentucky Department of Obstetrics and Gynecology Andrology Laboratory. MAIN OUTCOME MEASURES: Manual and computer-assisted measurements of sperm motility and motion characteristics. RESULTS: Gossypol inhibited sperm motility, which could be reversed partially by increasing cAMP. CONCLUSION: Gossypol exposure in vitro adversely affects sperm motility in a dose- and time-dependent manner by a cAMP-dependent mechanism.

PIP: The antimotility effect of gossypol on human sperm has been documented. Gossypol is commonly derived from cottonseed oil. The exact mechanism by which this effect occurs is unknown. This paper reports research on the effects of gossypol on sperm motility. Elucidation of the site(s) of action and whether the effects are reversible are also discussed. Fresh human sperm was collected, centrifuged into pellets of equal numbers, washed, and then either overlayered with Ham's F-10 media or treated with gossypol. Gossypol solutions contained 10, 12.5, 20, 25 or 50 mcg/mcl of gossypol. These concentrations were used in measuring the sperm dose-response. Forskolin, theophylline, or cyclic adenosine monophosphate (cAMP) was added to gossypol-treated sperm and motility levels were measured. Motility measurements were conducted manually or via computer-assisted readings of sperm motion characteristics and their actual ability to move. Antimotility effects of gossypol on sperm are related to both dose and exposure time. This study supports the hypothesis that gossypol inhibits cAMP formation. At lower doses of gossypol (10 mcg/mcl), both theophylline and forskolin reversed gossypol's antimotility effect. At higher concentrations (20 mcg/mcl), the effect of gossypol appeared to be rapid and was irreversible. This latter finding has implications for its use as a vaginal contraceptive agent.

Immunomodulation of rat endometriotic implant growth and protein production.

PROBLEM: The immune system has been implicated in the pathophysiology of endometriosis. To determine if modulation of the immune system influences endometriotic implant growth and protein production, the following experiment was conducted. METHOD: Female rats with surgically induced endometriosis were treated with either the immunosuppressive agent pentoxifylline (Pent; 5 mg/kg BW; N = 16) or a vehicle (N = 16) for 7 consecutive days, then killed. Twenty-four hours before death, 8 animals from each group were injected intraperitoneally with the immunostimulatory agent lipopolysaccharide (LPS; 200 micrograms/kg BW). At death, endometriotic implants were measured and protein production assessed by two-dimensional polyacrylamide gel electrophoresis. RESULT: Pentoxifylline significantly (P < 0.001) reduced endometriotic implant size (2.15 +/- 0.65 mm2 vs. 17.13 +/- 1.98 mm2) whereas LPS was without effect (18.32 +/- 2.57 mm2 vs. 17.13 +/- 1.98 mm2). Pentoxifylline also suppressed production of a portion of the proteins that comprise the implant specific group of proteins, ENDO-2, whereas LPS induced the production of two additional ENDO-2 proteins. CONCLUSION: Immunomodulatory agents can modulate rat endometriotic implant growth and production of implant-specific proteins.

Detection of a unique 32-kd protein in the peritoneal fluid of women with endometriosis.

OBJECTIVE: To test the hypothesis that endometriotic tissue secretes endometriotic-specific proteins into the peritoneal fluid (PF) of women with endometriosis. DESIGN: A prospective design was utilized in this study. SETTING: Tertiary care, university-based center and reproductive endocrinology laboratory. PARTICIPANTS: Women of reproductive age who were infertile with endometriosis (n = 19), as well as without endometriosis (n = 7), and fertile women undergoing tubal ligation (n = 6). INTERVENTIONS: Collection of PF fluid via laparoscopy. MAIN OUTCOME MEASURES: Peritoneal fluid proteins were isolated and assessed by two-dimensional polyacrylamide gel electrophoresis. RESULTS: Two-dimensional electrophoresis of PF proteins isolated a group of proteins (M(r) = 32 to 40 kd, pI = 4.5 to 5.2) in all PF samples that was similar to the rat endometriotic implant-specific protein, Endo-1. This group of proteins consisted of 5 to 12 individual proteins with endometriosis PF containing a significantly higher number of proteins (median = 11) compared with either PF from infertile women without endometriosis (median = 8) or from women undergoing tubal ligation (median = 7). In addition, one protein (M(r) = 32 kd, pI = 5.8), termed EPF-32, was detected predominantly (18 of 19 samples analyzed) in PF from women with endometriosis. This protein was also detected in PF from infertile women without endometriosis (2 of 7 samples) but not in the PF of fertile women undergoing tubal ligation (0 of 6 samples). The appearance of this protein was not associated with the severity of endometriosis. CONCLUSION: It is concluded from this study that PF from women with endometriosis predominantly contains a 32-kd protein (EPF-32) compared with the PF of women without the disease. The role of EPF-32 in the pathophysiology of endometriosis is not established but this protein may function as a diagnostic marker for endometriosis.

The presence of luteinized unruptured follicle syndrome and altered folliculogenesis in rats with surgically induced endometriosis.

OBJECTIVE: Our purpose was to determine in the rat model whether endometriosis could influence ovarian function by altering oocyte release or folliculogenesis. STUDY DESIGN: We histologically examined the ovaries of reproductively cycling rats with (n = 16) and without (n = 10) surgically induced endometriosis. The rats in these two groups were further subdivided into unilaterally ovariectomized or ovarian-intact groups. Serial sections of ovaries were examined, and follicular development and frequency of luteinized unruptured follicles were determined. RESULTS: A significant tenfold increase in the number of luteinized unruptured follicles was observed in the ovaries from rats with endometriosis (2.7 per rat) compared with unoperated and sham-operated control groups (overall mean 0.26 per rat, p < 0.05). Additionally, ovaries from unilateral ovariectomized animals with endometriosis contained four times as many luteinized unruptured follicles (four per rat) as did the ovaries from bilaterally ovarian-intact rats with endometriosis (1.40 per rat, p < 0.01). Fewer follicles were present in rats with endometriosis (180 follicles per ovary) than in control rats (231 follicles per ovary, p < 0.05). CONCLUSION: In the rat model the presence of ectopic endometrium is associated with an increased frequency of luteinized unruptured follicles and altered follicular development.

Polypeptides synthesized and released by rat ectopic uterine implants differ from those of the uterus in culture.

Unique endometriosis-specific secretory proteins would be of paramount importance as noninvasive markers for diagnosis and evaluation of therapeutic approaches for endometriosis. Furthermore, identification of endometriosis-specific secretory proteins may be an important step towards understanding the pathophysiology of endometriosis-associated pain and infertility. Therefore this study was designed to assess protein synthesis and secretion by ectopic uterine implants from steroid-treated and reproductively cyclic rats with surgically induced endometriosis. Uteri, ectopic uterine implants, and control tissues were incubated in L-[35S]methionine or D-[6-3H]glucosamine for 0-24 h and 24-48 h. De novo-synthesized proteins released into the culture media were identified using two-dimensional SDS-PAGE, fluorography, and computer-assisted image analysis. Two distinct groups of ectopic uterine implant proteins were identified: ENDO I (M(r) 40,000-50,000; pI 4.0-5.2) and ENDO II (M(r) 28,000-32,000; pI 7.5-9.0) were produced by ectopic uterine implants and not the uteri. A third group of proteins, previously identified in culture media of the uteri from progesterone-treated rats and called PUP-1 (M(r) 70,000; pI 5.7), was synthesized and secreted by ectopic uterine implants 24-48 h later than in parallel uterine cultures. The detection of ectopic uterine implant proteins suggests biochemical characteristics of the ectopic tissue that may be used to develop unique markers for endometriosis. Furthermore, the delayed synthesis and secretion of the uterine protein PUP-1 by the ectopic uterine implants illustrates yet another example of the asynchronous behavior of these two tissues, which may be related to the etiology or pathophysiology of the disease.

Development and characterization of a polyclonal antiserum-based radioimmunoassay for dog osteocalcin.

Determination of the serum concentration of the protein osteocalcin (OC) is useful for the noninvasive evaluation of bone metabolism. Because the dog is an excellent experimental model for the study of bone, we produced and characterized a polyclonal antiserum specific for dog OC and used it to develop a radioimmunoassay (RIA) for the measurement of the concentration of this protein in dog serum. The antiserum expresses higher affinity for Ca(2+)-bound than for Ca(2+)-free OC (B50 at 10(-5) versus 2 x 10(-4) dilution). Also, in the presence of Ca2+ affinity is higher for the carboxylated than for the decarboxylated form of the protein, and under Ca(2+)-free conditions the affinity is equal for the two forms. The study of peptide fragments of OC demonstrates competitive binding of the peptide comprising amino acids 20-44 but not of other fragments; this suggests that the antigenic epitope of dog OC is located in the midmolecular region of the protein. The RIA displays excellent sensitivity for the measurement of OC in blood (detection limit 0.31 ng/ml), with intraassay and interassay variations of 4.6 and 6.8%, respectively. Analysis of gel chromatography fractions of normal dog serum shows that greater than 90% of the antigenic material coelutes with purified radiolabeled dog OC. Test of parallelism reveals lack of interference of serum constituents with the binding assay. The antiserum displays limited species specificity since it cross-reacts with human OC, but not with the protein from rodents.(ABSTRACT TRUNCATED AT 250 WORDS)

Embryonic losses after 10-week administration of cobalt to male mice.

Cobalt toxicity was evaluated in the dominant lethal assay (DLA) to determine whether the detrimental effects of cobalt on spermatozoa would have an impact on offspring. Male B6C3F1 mice were treated with cobaltous chloride (400 ppm Co) for 10 weeks and mated. Neither the stage nor rate of development in vitro of 2-cell embryos to blastocyst from cobalt-treated males was affected. There was an increase in preimplantation losses and a decrease in total and live births, but no change in postimplantation losses from litters at day 19 of gestation. Fertility of the males was maintained during the 10-week cobalt treatment period, decreased during the DLA, and recovered over the next 6 weeks. Sperm parameters at the end of DLA and the recovery period showed that cobalt decreased all parameters measured at 12 weeks, but these parameters, except concentration, recovered to control levels by 18 weeks. Tissue concentrations of cobalt measured by atomic absorption analysis were increased in liver, kidney, testis, and epididymis after 12 weeks of cobalt treatment. We conclude that cobalt affected preimplantation losses in the DLA by compromising the fertility of treated males.

Enoximone. A review of its pharmacological properties and therapeutic potential.

Enoximone is an imidazolone derivative currently undergoing trials in patients with congestive heart failure refractory to conventional therapy. It is a phosphodiesterase inhibitor with both positive inotropic and vasodilator properties, and is active by both oral and intravenous routes of administration. While pharmacodynamic studies have documented beneficial haemodynamic effects after short term oral administration, and objective and subjective improvement relative to placebo during some short term trials, its clinical efficacy during continuous longer term therapy remains uncertain. Enoximone is of potential benefit as an adjunct in short term management of patients with end-stage cardiac failure awaiting cardiac transplantation. In the usually small studies reported to date enoximone was generally better tolerated at oral dosages of less than 2 mg/kg than at higher dosages. Thus, while its pharmacodynamic profile holds potential promise in a difficult therapeutic area, the long term clinical efficacy and tolerability of enoximone remain yet to be determined in controlled trials of adequate size and duration.

Detection of a progesterone-induced secretory protein synthesized by the uteri but not the endometriotic implants of rats with induced endometriosis.

Steroids modulate the secretory activity of the uterus, but little is known of their effect on ectopic endometrium protein synthesis and secretion. We utilized two-dimensional electrophoresis to visualize proteins produced by the uteri and endometriotic implants of both steroid-treated and reproductively cyclic rats with and without surgically induced endometriosis. Of the greater than 300 proteins visualized, only the uterine cultures from progesterone (P)-stimulated, estrogen-suppressed rats contained a distinctive glycoprotein (P-induced uterine protein-1; molecular weight [Mr] 70,000; isoelectric point [pI] 5.7). This protein was not detected in any of the endometriotic implant cultures. Progesterone-induced uterine protein-1 could play a role in luteal or endometrial physiology and may be valuable in assessing endometrial function. The aberrant secretory behavior of the ectopic endometrium suggests a possible involvement in the reproductive dysfunction associated with endometriosis.

Piracetam. An overview of its pharmacological properties and a review of its therapeutic use in senile cognitive disorders.

Piracetam is the first of the so-called 'nootropic' drugs, a unique class of drugs which affect mental function. In animal models and in healthy volunteers, the drug improves the efficiency of the higher telencephalic functions of the brain involved in cognitive processes such as learning and memory. The pharmacology of piracetam is unusual because it protects against various physical and chemical insults applied to the brain. It facilitates learning and memory in healthy animals and in animals whose brain function has been compromised, and it enhances interhemispheric transfer of information via callosal transmission. At the same time, even in relatively high dosages it is devoid of any sedative, analeptic or autonomic activities. How piracetam exerts its effects on memory disorders is still under investigation, although among other proposed mechanisms of action it is thought to facilitate central nervous system efficiency of cholinergic neurotransmission. Results from trials involving elderly patients with senile cognitive disorders have been equivocal, as have the results obtained when piracetam has been combined with acetylcholine precursors. Piracetam seems to be almost completely devoid of adverse effects, and is extremely well tolerated. In conclusion, opinion is divided as to the benefits of piracetam in the treatment of senile cognitive decline. Although double-blind studies in the elderly have produced mixed results, some such trials (particularly those involving larger numbers of patients) have reported favourable findings, thus offering some reason for cautious optimism in a notoriously difficult area of therapeutics. However, further investigations of piracetam alone and in combination therapy are required before any absolute conclusions can be drawn.

Relationships of serum estriol, cortisol and albumin concentrations with pig weight at 110 days of gestation and at birth.

In polytocous species, animals with reduced birth weights are associated with reduced neonatal survival, which may be related to substandard placental function. At 110 days of gestation (n = 84) and at birth (approximately 114 days, n = 193), fetal pigs were bled and weighed, so that indexes of placental function (estriol), fetal stress (cortisol), and fetal growth (albumin) could be related to fetal development. Concentration of estriol at birth or 110 days of gestation was not linearly related to weight of pig. Pig serum albumin was linearly related to pig weight and increased as body weight increased (p less than 0.05). In blood from pigs sampled at birth, cortisol concentrations significantly decreased as body weight increased (p less than 0.05).

Spontaneous and steroid-induced recurrence of endometriosis after suppression by a gonadotropin-releasing hormone antagonist in the rat.

Recurrent endometriosis in women is difficult to study because of the ethical consideration of performing repeated surgeries. Previously in the rat model we described therapeutic regression of endometriosis with the gonadotropin-releasing hormone antagonist antide. Presently we report the spontaneous and steroid-induced recurrence of endometriosis after withdrawal from antide therapy. Rats with endometriosis received antide or vehicle on days 0 (proestrus), 3, 6, and 9 and were killed on days 0, 6, 12, 18, 24, 30, and 42 (n = 4 antide-treated and 4 vehicle-treated rats killed per day). Additional antide-treated rats (n = 4 per treatment) received estrogen, progesterone, both estrogen and progesterone, cholesterol, and no steroid on day 9 and were killed on day 12. Antide significantly suppressed endometriotic implant size on days 12, 18, and 24. However, implant size spontaneously returned to pretreatment values by day 30. Administration of steroids on day 9 elicited regrowth of antide-suppressed endometriosis (estrogen plus progesterone greater than estrogen, progesterone, or cholesterol greater than no steroid) by day 12. This resilience of endometriosis offers an explanation for treatment failure and recurrence of the disease in women.

Follicular atresia and infertility in rats treated with a gonadotropin-releasing hormone antagonist.

Adverse effects of the GnRH antagonist Antide on folliculogenesis and fertility were noted when we were evaluating the therapeutic value of Antide on endometriosis in a rat model. Cyclic rats with (n = 56) and without (n = 18) surgically induced endometriosis received Antide (2 mg/kg) or vehicle at noon on days 0 (proestrus), 3, 6, and 9. Rats were killed at noon on days 0, 6, 12, 18, 24, 30, 42, and 165. The number of antral follicles and the number of atretic antral follicles evaluated did not differ (P greater than 0.05) between endometriosis and control rats. Antide-treated rats had more (P less than 0.05 ) atretic antral follicles (64.7%) than vehicle-treated rats (15.3%). Antide-treated rat ovaries contained fewer corpora lutea than those of vehicle-treated rats on days 6, 12, and 18. No corpora lutea were found in Antide-treated rat ovaries after day 18. The abnormal ovarian morphology of the Antide-treated rats persisted for the duration of the project (165 days). Fertility (rats without endometriosis) was assessed by mating vehicle-and Antide-treated rats at spontaneous proestrus (n = 8) for eight posttreatment cycles as well as after follicular stimulation (n = 2). All vehicle-treated and no Antide-treated rats became pregnant. No oocytes were found in the oviducts of Antide-treated rats after eight cycles, indicating that ovulation had not occurred. The serum FSH and estradiol concentrations in the rats treated with Antide were lower (P less than 0.05) on days 6, 12, and 18, but rose to values equal to (days 24 and 30) or greater than (day 42) those in vehicle-treated rats. Serum progesterone levels in rats treated with Antide were lower (P less than 0.05) than those in vehicle-treated rats on all days tested. In conclusion, at a dosage sufficient to suppress reproductive cyclicity (and elicit the regression of endometriosis), Antide also caused long term follicular atresia and infertility.

Experience with a physiology workshop for high school and college teachers.

Science education in the United States at all academic levels is widely perceived to need direct assistance from professional scientists. The current dearth of quality applicants from this country to medical and graduate schools suggests that our existing undergraduate and high school science curriculum is failing to provide the necessary stimulus for gifted students to seek careers in the health sciences. Recognizing the need to become more directly helpful to high school and college science teachers, members of the faculty of the Department of Physiology and Biophysics at the University of Kentucky College of Medicine held a 5.5-day Physiology Summer Workshop during June, 1989. Participants included 25 college teachers from Kentucky and 5 other states plus 22 Kentucky high school teachers. The presence of the two levels of educators provided communication about curricular concerns that would be best addressed by mutual action and/or interaction. Each day's activities included morning lectures on selected aspects of organ system and cellular physiology, a series on integrative physiology, and afternoon laboratory sessions. The laboratory setting allowed the instructor to expand on principles covered in lecture as well as provided the opportunity for in-depth discussion. A selection of evening sessions was presented on 1) grants available for research projects, 2) obtaining funds for laboratory equipment, and 3) graduate education in physiology.

Characterization of a computerized semen analysis system.

In the present studies we have evaluated the optimal operating conditions for the Hamilton-Thorn HTM-2000 computerized semen analyzer (Hamilton-Thorn, Danvers, MA). The best reproducibility in measurement of sperm concentration was obtained using 20 frames acquired at 19 frames/s. The measurement of sperm concentration was not adversely affected by the number of fields analyzed. The intrasample and intersample coefficients of variation for sperm concentration were 9.5% and 25.5%; sperm motility, 18.4% and 28.9%; lateral head displacement, 16.5% and 19.9%; path velocity, 6.8% and 13.9%; progressive velocity, 4.5% and 9.9%; and linear index, 2.5% and 4.2%; respectively. These differences suggest that sampling error has a significant influence on the reliability of sperm evaluation. The precision and rapidity of the HTM-2000 compares favorably with data previously reported from other systems available for clinical semen analysis.

Plasminogen activator and plasminogen activator inhibitor in human preovulatory follicular fluid.

To evaluate the roles of plasminogen activator (PA) and PA inhibitor (PAI) in human ovulation, we obtained follicular fluid and granulosa cells from individual preovulatory follicles of patients undergoing gamete intrafallopian tube transfer. The follicular fluid samples (n = 25) were analyzed for total tissue-type PA antigen, PA enzyme activity by fibrin autography, PAI activity, PAI type 1 (PAI-1) antigen, and PAI-1 mRNA. The follicular fluid of preovulatory follicles contained low levels of total tissue-type PA antigen (less than 1 ng/mL). Fibrin autography experiments indicated little or no detectable PA activity associated with free or unbound PA. The results of the PAI activity assay and PAI-1 antigen determination support the concept of a relative abundance of PAI compared with PA. Hybridization analysis was used to measure the relative amounts of granulosa cell PAI-1 mRNA. The levels of PAI-1 mRNA correlate with follicular fluid PAI concentrations in individual follicles. Taken together, these results support the idea that there is very little free, or active, PA in follicular fluid of human preovulatory follicles, but there is an abundance of PAI. Furthermore, PAI-1 produced by the granulosa cells may represent a major form of PAI in follicular fluid.

Identification and characterization of metalloproteinase inhibitor activity in human ovarian follicular fluid.

Metalloproteinase inhibitors regulate collagenase activity in the extracellular matrix. To assess the role of metalloproteinase inhibitors in the ovulatory process, inhibitor activity was examined in human follicular fluid collected 2-4 h before ovulation. The relationship between inhibitor activity and steroid content was determined, and the inhibitors were partially purified and characterized. Inhibitory activity in follicular fluid (n = 25) correlated with both follicular estradiol (P less than 0.001) and progesterone (P less than 0.02) concentrations per follicle. Chromatographic separation of the follicular fluid on Sepharose 6B isolated two peaks of inhibitory activity. The inhibitor from the small mol wt (Mr) peak shared many of the properties of tissue inhibitors of metalloproteinase. It was stable in response to heat (60 C) and methylamine (200 mM), and was destroyed by reduction and alkylation, a procedure reported to destroy previously characterized inhibitors. Partial purification by affinity and ion exchange chromatography demonstrated the inhibitor to be a glycoprotein with an approximate Mr of 28-29K. The large Mr inhibitor had an approximate size of 700K and exhibited many of the characteristics of alpha 2-macroglobulin, a serum-derived metalloproteinase inhibitor. It was sensitive to heat, methylamine, and reduction and alkylation. Thus, follicular fluid contains metalloproteinase inhibitor activity that is steroid related and may be hormonally regulated. Ovarian metalloproteinase inhibitors may act to regulate connective tissue remodeling during follicular rupture.