A new radioimmunoassay for human chorionic gonadotropin using monoclonal antibody

A new radioimmunoassay (RIA) for human Chorionic Gonadotropin (hCG) was developed using murine monoclonal antibody to the beta-subunit of hCG (beta-hCG). The IgG fraction of the monoclonal antibody which did not react with 125I-beta-hCG was purified from hybridoma ascites, and covalently coupled to Sepharose 4B. This solid-phase antibody was incubated with standard hCG or serum sampled for 48 hours. The reaction medium was then removed by centrifugation and 125I-beta-hCG and anti-beta-hCG rabbit polyclonal antibody were added to the precipitate. The alcohol precipitation method was used for separating "bound" and "free" forms in the second reaction. The sensitivity for hCG in this assay system was 0.5 mIU/ml serum and the cross-reactivity with human Luteinizing Hormone (hLH) was 0.4%. This assay system was shown to be clinically applicable. Serial serum samples from two patients with trophoblastic disease were assayed and minute amounts of hCG, which could not be determined by conventional assay methods, could be assayed by this new RIA.     

Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous subcutaneous infusion of human menopausal gonadotropin in anovulatory women with decreased FSH/LH ratio and androgen excess

Ten women of polycystic ovarian-type (PCO-type) anovulation, having a decreased ratio of FSH to LH and androgen excess, resistant to the previous clomiphene, bromocriptine and/or daily im injections of human menopausal gonadotropine (hMG), were treated with continuous sc infusion of 150 IU/day hMG. The treatment was initiated on cycle day 2-5 and continued until the dominant follicle reached 20 mm or more in diameter, when an im bolus of 10,000 IU human chorionic gonadotropin was given. The treatment elevated the geometric mean of pretreatment serum FSH (8.6 mIU/ml) to 15.9 mIU/ml (p less than 0.001), while serum LH decreased from 29.4 mIU/ml to 20.7 mIU/ml (p less than 0.01). This resulted in a highly significant increase in the FSH/LH ratio from 0.29 to 0.77 (p less than 0.0001). Follicle enlargement was demonstrated in 13 of the 14 treatment cycles, 12 of which were ovulatory. Pregnancy ensued in 4 cases, 1 of which was a quadruplet pregnancy. Continuous infusion of hMG was indicated as an effective way of inducing ovulation in PCO-type anovulation resistant to conventional methods of ovulation induction.     

Effect of fetal sex on maternal and fetal human chorionic gonadotropin levels and comparison of their levels in paired umbilical arteries and veins

There were discordant results regarding the effect of fetal sex on human chorionic gonadotropin (hCG) concentrations in maternal or fetal circulation and regarding whether the levels in umbilical arteries are equal to those in umbilical veins. Totally, 188 singleton pregnancies at 36 to 42 weeks of gestation without any obstetrical or medical complication were studied. The hCG levels were measured by radioimmunoassay specific for hCG using Sb3 antibody raised against beta-subunit of hCG. The maternal ages and parities between those who gave birth to a male or a female baby were not different statistically. The birth weights between male and female babies were also not different. The serum hCG levels had a wide range in maternal circulation (200-75,200 mIU/ml) and their distribution was positively skewed. The mean value (geometric mean, G.M.) in maternal circulation for those who carried a female fetus (11,500 mIU/ml) was significantly higher than that for those carrying a male fetus (6,470 mIU/ml) (P less than 0.001, Student's t-test). The hCG concentrations in umbilical veins of female fetuses were also higher than in those of male fetuses (G.M., 26.8 vs 19.5 mIU/ml, P less than 0.01, Student's t-test). Umbilical arterial hCG levels (G.M., 10.05 mIU/ml) were statistically not different from umbilical venous levels (G.M., 10.92 mIU/ml) (paired t-test).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of culture medium,serum type,and various concentrations of follicle-stimulating hormone on porcine preantral follicular development and antrum formation in vitro

Developing a culture system for preantral follicles has important biotechnological implications due to the potential to produce large number of oocytes for embryo production and transfer. As an initial step toward accomplishing this long-term goal, a study was conducted to determine the effects of culture medium, serum type, and different concentrations of FSH on preantral follicular development in vitro. Specific endpoints included follicular growth rate, antrum formation, recovery rate of cumulus cell-oocyte complexes (COCs) from follicles, and oocyte meiotic competence. Compared with the North Carolina State University medium 23 (NCSU23), preantral follicles cultured in TCM199 medium for 4 days grew faster (P < 0.02). However, more follicles cultured in NCSU23 differentiated to form an antrum than in TCM199 (P < 0.01). For this reason, NCSU23 was chosen to investigate the role of FSH and serum type in regulating preantral follicular growth. Compared with the 0 mIU/ml FSH control, addition of 2 mIU/ml FSH to the medium stimulated follicular growth and antrum formation and suppressed apoptosis of granulosa cells (P < 0.05), supporting the essential role of FSH in preantral follicular growth and development. Another experiment compared fetal calf serum (FCS) with prepubertal gilt serum (PGS) and studied different concentrations of FSH in the culture medium (0.5, 1, and 2 mIU/ml). The best follicular growth rate was obtained with 2 mIU/ml compared with 0.5 or 1 mIU/ml FSH. Compared with PGS, FCS supplementation increased the cumulative percentage of antral follicles and COC recovery rate (P < 0.04). None of the oocytes recovered from any of these experiments reached metaphase II stage after maturation in vitro. In summary, culture medium, serum type, and FSH concentration in the medium interacted to affect follicular growth and antrum formation in vitro. These results suggest that a longer term culture of preantral follicles (>4 days) may be needed to produce oocytes capable of undergoing meiosis in vitro.     

Reagentless amperometric immunosensor for human chorionic gonadotrophin based on direct electrochemistry of horseradish peroxidase

A novel amperometric immunosensor for determination of human serum chorionic gonadotrophin (HCG) was constructed by immobilization of HCG with titania sol-gel on a glassy carbon electrode and the direct electrochemistry of horseradish peroxidase (HRP) labeled to HCG antibody (HRP-anti-HCG). The morphologies of the HCG membrane were characterized to be chemically clean, porous and homogeneous. HRP-anti-HCG was functionally conjugated with the immobilized HCG after incubation in phosphate buffer (PBS) containing HRP-anti-HCG. A direct electron transfer of HRP with a rate constant of 1.35+/-0.40 s(-1) was observed at the HRP-anti-HCG-HCG/titania sol-gel membrane modified electrode in 0.1 M PBS pH 7.0. With a competitive mechanism the differential pulse voltammetric peak current of the immobilized HRP decreased linearly with an increasing HCG concentration from 2.5 to 12.5 mIU/ml in the incubation solution. The HCG immunosensor showed a detection limit of 1.4 mIU/ml, a good accuracy and acceptable precision and reproducibility with an intra-assay CV of 4.7% at 5.0 mIU/ml and an inter-assay precision of 8.1% obtained at 10 mIU/ml. The biosensor displayed a good stability in a storage period of 30 days.     

Production of antibodies specific to human chorionic gonadotropin in mice immunized against its chemical analogs

Mice immunized against DS₅-hCG-Β and DS₆-hCG-Β, chemical analogs of Β-subunit of human choriogonadotropin (hCG-Β) in which 5 and 6 disulphide bonds respectively were reduced and alkylated, were found to produce antibodies specific to hCG without significant crossreactivity with human lutropin (hLH) as tested in a radioimmunoassay. In contrast, mice immunized against the native hCG-Β subunit produced hLH crossreacting antibodies. While the anti-DS5, DS6-hCG-Β serum was capable of selectively blocking the binding of [¹²⁵I]-hCG to rat testicular LH/hCG receptors, it failed to inhibit the binding of [₁₂₅I]-hLH to the same receptors. The radioimmunoassay for hCG using the mouse anti-DS₅, DS₆-hCG-Β serum was not as sensitive as that employing rabbit anti-DS₅, DS₆-hCG-Β serum. The minimal detection limit was 5 ng/ml for the mouse antibody as compared to 1 ng/ml for the rabbit antibody. Supported by U.S. Public Health Service Grant HD 08766 to OPB. An erratum to this article is available at .     

抗—HBs国际单位RIA一点法定量测定与计算方法的探讨

在Radio-immuno Assay(RIA)试验测抗－HBs国际单位的简易定量与计算中,采用RIA一点法测定,以分析表达式mIU/ml＝SC（mIU/ml)[exp(0.69315S-NC/SC-NC-1]进行结果计算。此式可以常用对数式表示为mIU/ml=SC(mIU/ml)[lg^-1(0.30103S-NC/SC-NC-1],当（S－NC）在0．3－1．1界线内时,其计算结果的相对误差小于6％,是目前误差最小的简易计算方法。同时还推算求出Holliger公式的阳性对照的最适含量为124．26mIU／ml,Richardson公式的阳性对照为125mIU/ml,中国药品生物制品检定所公式的阳性对照为92．5mIU/ml。     

Presence of positive feedback in males with Klinefelter's syndrome

This study examined the effect of 17 beta-estradiol (E2) on basal and luteinizing hormone (LH)-releasing hormone (LHRH)-stimulated gonadotropin secretion in 9 patients with Klinefelter's syndrome. Intramuscular injection of E2 (10 micrograms/kg/day during 5 days) induced a rapid decrease in follicle-stimulating hormone (FSH) and LH levels. The maximum suppression was observed on day 7 (D7) for FSH [median 9.7 mIU/ml (range 4.6-37.8) vs. 21.7 mIU/ml (range 12.2-56.9)] and on D2 for LH [median 13.6 mIU/ml (range 6.8-25.2) vs. 21.2 mIU/ml (range 13-54.7)]. E2 concentrations rose and reached their peak values on D3 [median 723 pmol/l (range 517-1,247.8) vs. 110.1 pmol/l (range 68.6-227.5) on D0]. These changes were followed by a subsequent rise in LH on D4 [36.7 mIU/ml (range 19.4-77.7)]. LH response to LHRH was higher during E2 treatment: median value of absolute peaks: 156.3 mIU/ml (range 56.7-188.6) on D4 vs. 64 mIU/ml (range 38.9-131) on DO. These results demonstrate the presence of a positive feedback in patients with Klinefelter's syndrome.     

Neuroimmunomodulation of the immune response by the endocrine system: effect of luteinizing hormone (LH) on the proliferative response of lymphocytes

Proliferative response of splenic lymphocytes from adult (2 months old) male mice to human luteotrophic hormone (hLH) was studied in vitro. hLH induced lymphoblastic transformation at a concentration of 0.1 micrograms/ml or higher. This hormone potentiated the mitogen activities of concanavalin A (ConA) and phytohemagglutinin (PHA) for T cells and of pokweed mitogen for B and T cells. The data suggest that peripherally circulating LH may regulate a certain function of lymphoid cells.     

血清P、β-HCG联合检测预测和治疗先兆流产的临床研究

目的：探讨血清孕酮与B-HCG联合检测在预测早期先兆流产预后及其治疗的临床价值。方法：对340例早期先兆流产患者血清P与B-HCG进行检测,并与追踪到的妊娠结局进行分析。结集：血清P值〉25ng／ml、血β-HCG〉50mIU／ml患者66例占19．4％,经绝对卧床休息,未用药治疗,均胚胎发育正常;血清P值在15．94-25ng／ml、血β-HCG10-50mlU／ml患者170例占50％,经口服黄体酮与HCG针保胎治疗后均胚胎发育正常;血清P值〈15．94ng／ml、血β-HCG〈10mIU／ml患者78例占22．9％,终止妊娠者均可见清除宫内组织物中几乎不见新鲜绒毛并伴有不同程度的陈旧性出血;血清孕酮与β-HCG上升不同步患者26例占7．6%,均最终难免流产。结论：联合检测血清孕酮与β-HCG可以预测先兆流产患者妊娠结局,对于指导治疗具有重要的临床价值,既避免不必要的药物干预及经济负担,又能起到提高保胎治疗的成功率。绒毛膜促性腺激素配伍口服天然黄体酮治疗由黄体功能不全引发的早期先兆流产,均能改善先兆流产患者的预后,且对母儿无不良影响,安全有效。     

Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.

Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...     

Progesterone production by dispersed luteal cells of non-pregnant cows: effects of oxytocin and oestradiol

The effects of oxytocin and oestradiol on progesterone production by dispersed luteal cells of non-pregnant cows were studied. In acute incubation (3 h), oxytocin, at a concentration of 800 mIU/ml, significantly inhibited the production of progesterone induced by HCG (10 IU/ml). Suppression of basal progesterone production was evident in some corpora lutea. Lower oxytocin concentrations (4 and 40 mIU/ml) had no effect. At a concentration of 400 mIU/ml, oxytocin may be inhibitory to basal and HCG-induced progesterone production. Oestradiol (1 μkg/ml) had no effect on basal progesterone production but may suppress the production of progesterone induced by HCG. However, incubation with oxytocin (400 mIU/ml) plus oestradiol (1 μg/ml) resulted in a significant inhibition of HCG-induced progesterone production. These data provide evidence for an inhibitory effect of oxytocin on the corpus luteum of non-pregnant cows. Oestradiol may interact with oxytocin to inhibit the bovine corpus luteum function.     

Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro

The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 μg/ml human follicle-stimulating hormone (hFSH), (2) 10 μg/ml human luteinizing hormone (hLH), (3) 1.0 μg/ml hFSH and 10 μg/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 ± 1.7 hr; hLH: 10.1 ± 1.8 hr; both gonadotropins: 8.8 ± 1.1 hr) when compared to controls (17.4 ± 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 ± 1.2 hr; hLH: 35.1 ± 1.4 hr; both gonadotropins: 35.2 ± 1.3 hr; no gonadotropins: 34.1 ± 1.2 hr). The mean interval to extrusion of PB1 was 34.1 ± 0.6 hr. In conclusion, GVB and PB1 extrusions appear to be, in part, independently regulated events in macaque oocytes matured in vitro since the timing of PB1 extrusion is not tightly coupled with the onset of GVB. Although the developmental potential of oocytes may be enhanced by gonadotropins, alternative approaches must be developed to improve the poor competence of oocytes from nonstimulated monkeys to mature in vitro. © 1994 Wiley-Liss, Inc.     

Luteinizing hormone, a reproductive regulator that modulates the processing of amyloid-beta precursor protein and amyloid-beta deposition

Hormonal changes associated with the dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis following menopause/andropause have been implicated in the pathogenesis of Alzheimer's disease (AD). Experimental support for this has come from studies demonstrating an increase in amyloid-beta (Abeta) deposition following ovariectomy/castration. Because sex steroids and gonadotropins are both part of the HPG feedback loop, any loss in sex steroids results in a proportionate increase in gonadotropins. To assess whether Abeta generation was due to the loss of serum 17beta-estradiol or to the up-regulation of serum gonadotropins, we treated C57Bl/6J mice with the anti-gonadotropin leuprolide acetate, which suppresses both sex steroids and gonadotropins. Leuprolide acetate treatment resulted in a 3.5-fold (p < 0.0001) and a 1.5-fold (p < 0.024) reduction in total brain Abeta1-42 and Abeta1-40 concentrations, respectively, after 8 weeks of treatment. To further explore the role of gonadotropins in promoting amyloidogenesis, M17 neuroblastoma cells were treated with the gonadotropin luteinizing hormone (LH) at concentrations equivalent to early adulthood (10 mIU/ml) or post-menopause/andropause (30 mIU/ml). LH did not alter amyloid-beta precursor protein (AbetaPP) expression but did alter AbetaPP processing toward the amyloidogenic pathway as evidenced by increased secretion and insolubility of Abeta, decreased alphaAbetaPP secretion, and increased AbetaPP-C99 levels. These results suggest the marked increases in serum LH following menopause/andropause as a physiologically relevant signal that could promote Abeta secretion and deposition in the aging brain. Suppression of the age-related increase in serum gonadotropins using anti-gonadotropin agents may represent a novel therapeutic strategy for AD.     

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.     

Effects of gonadotrophins, growth hormone, and activin A on enzymatically isolated follicle growth, oocyte chromatin organization, and steroid secretion

So far, standard follicle culture systems can produce blastocyst from less than 40% of the in vitro matured oocytes compared to over 70% in the in vivo counterpart. Because the capacity for embryonic development is strictly associated with the terminal stage of oocyte growth, the nuclear maturity status of the in vitro grown oocyte was the subject of this study. Mouse early preantral follicles (100-130 microm) and early antral follicles (170-200 microm) isolated enzymatically were cultured for 12 and 4 days, respectively, in a collagen-free dish. The serum-based media were supplemented with either 100 mIU/ml FSH (FSH only); 100 mIU/ml FSH + 10 mIU/ml LH (FSH-LH); 100 mIU/ml FSH + 1 mIU/ml GH (FSH-GH) or 100 mIU/ml FSH + 100 ng/ml activin A (FSH-AA). Follicle survival was highest in follicle stimulating hormone (FSH)-AA group in both cultured preantral (91.8%) and antral follicles (82.7%). Survival rates in the other groups ranged between 48% (FSH only, preantral follicle culture) and 78.7% (FSH only, antral follicle culture). Estradiol and progesterone were undetectable in medium lacking gonadotrophins while AA supplementation in synergy with FSH caused increased estradiol secretion and a simultaneously lowered progesterone secretion. Chromatin configuration of oocytes from surviving follicles at the end of culture revealed that there were twice more developmentally incompetent non-surrounded nucleolus (NSN) oocytes (>65%) than the competent surrounded nucleolus (SN) oocytes (<34%). We conclude that the present standard follicle culture system does not produce optimum proportion of developmentally competent oocytes.     

In vitro studies of prolactin inhibition of luteinizing hormone action on Leydig cells of rats and mice

Previous in vivo studies have shown that in male rabbits prolactin inhibits the testosterone production stimulated by luteinizing hormone (LH) or human chorionic gonadotropin (hCG). This inhibition has now been studied in vitro using both mouse and rat testicular interstitial cells. First, the dose response of human LH (hLH) stimulation of testosterone was studied in detail using testicular interstitial cells from both species. Next, a small but stimulatory dose of hLH was selected and extensive prolactin doses were studied in vitro. NIH B-6 (bovine) prolactin in varying doses was added to the interstitial cells 30 min prior to the addition of a constant dose of hLH. Under these circumstances prolactin inhibited LH action over a wide range of doses. In both species a biphasic dose-response curve existed: large doses of 100 to 1000 ng/ml produced less inhibition or augmented LH action, compared to smaller doses. Next, entire hLH dose-response curves were produced in the presence of three doses of prolactin (0.33, 33, and 1000 ng/ml) as well as in the absence of prolactin. The addition of prolactin shifted the hLH dose-response curve to the right and depressed the maximal response in comparison to the curve without prolactin. Finally, inhibitory doses of prolactin resulted in no detectable change in LH receptor number as estimated from Scatchard plots. It is concluded that prolactin inhibits LH action on interstitial cells as determined by rate of testosterone production except at very large doses of prolactin where LH action is less inhibited or augmented. The inhibitory action of prolactin in this in vitro interstitial cell assay was not accompanied by a decrease in LH receptor number. Thus, a postreceptor action is likely to be involved.     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.     

Evaluation of autoantibodies against oxidized LDL and antioxidant status in top soccer and basketball players after 4 months of competition

Antioxidant status and titers of autoantibodies against oxidized low-density lipoproteins (ox-LDL-Ab) were investigated in top soccer (S; n = 21, age 24.6 +/- 4.3 years) and basketball (B; n 3,000 mIU/ml) in ox-LDL-Ab were found in half the players (12S and 4B) with a maximum reaching 6000 mIU/ml (normal range: 200-600 mIU/ml), showing an in vivo LDL oxidation. There was no correlation between ox-LDL-Ab titers and chlolesterol, LDL cholesterol, or antioxidant levels. Nevertheless, plasma vitamin C concentration was lower in athletes having high levels of ox-LDL-Ab when compared with those with normal levels (8.49 +/- 3.14 mirogram/ml vs. 10.39 +/- 2.55 microgram/ml), but this difference was not statistically significant. In conclusion, our data suggest that potential atherogenic and cardiovascular risks as reflected by high titers in ox-LDL-Ab may exist in some top athletes despite a nonaltered antioxidant status.