Document gZwen15OzbO3nYERJZXyJVMV

BIOLOGY OF REPRODUCTION 41, 1055-1062 (1989) Effects of Lead on Luteal Function in Rhesus Monkeys1 PATRICIA A. FRANKS,3-4 NELLIE K- LAUGHLIN,3 DONALD I. DIERSCHKE,5 ROBERT E. BOWMAN,3-4 and PATRICIA A. MELLER5 NcolowPrirrwXe laboratoryf Environmental Toxicology Center,4 and Wisconsin Regional Primate Research Center5 l/nrversiiycfWisconsin "2 \MadisonfWisconsin 53715-1299jt. N 27040 ABSTRACT Exposure to leadin the workplace Or home environment has.been implicatedasa cause ofdecreasedfertility in women. In a previous study, aspart ofour effort to determine effects oflead in primates,female rhesus monkeys were exposed to lead acetate in drinking water(a=jO) or provided water with no added lead (m=7) for 33 mo. Lead, was administered at levels between2 and 8 mglkglday, with doses adjusted to keep blood lead values near a target of70 pgidl (observed mean iSEflf = 6S.9 6,54 \igldl). Blood leadconcentrations in control animals were < 10 yig/dl. No significant differences were detected between control and experimental animals in body weight, hematocrit, or general health. Female monkeys receiving lead exhibited longer and more variable menstrual cycles and shorter menstrualflow. In thepresent study, circulating amounts ofprogesterone (P4) were determined to evaluate lutealfunction during thefinal 7 mo oftreatment with lead. Several characteristics were altered os a result oflead treatment: circulating.amounts of?4 were reduced aS indicated by relative units ofarea under the concentration-time curve, maximal amounts ofP4 were reduced, and P4 levels were > 1 ngiml on fewer days. Therewere np significant differences between groups in mean percent qfanovulatory cycles. Therefore, although chronic treatment with the levels of lead used in this study did not prevent ovulation, luteal function was sup pressed. These results extendprevious observations ofadverse effects oflead on ovarian activity andfertility in monkeys, i INTRODUCTION Reproductive biologists are concerned with lead be cause of its toxic effects on the reproductive system. Lead has long been recognised as a toxicant (Hamilton and Hardy, 1983), and much effort has been devoted to reducing die use of lead in our daily lives. Neverthe less, exposure to lead is still prevalent in tile form of at mospheric lead from vehicular exhaust, lead solder in water pipes, and lead used in various industries. Lead has damaging effects on fertility, pregnancy, and fetal development in women living near or working in facto ries using lead (Rom, 1976; Hamilton and Hardy, Accepted August 1, 1989. Received February 24, 1988. _ _. , .SuPPOed by MB goats: RR 00167; ROl ES OIOSfcNarional Research ST?* Award T32 ES07015; Biomedical Research Grant; and EPA Grant 12- Contribution 217 from Environmental Toxicology Center, Univer- wy orwi. Madison, WI53706. Publication No. 29-015 from the Wisconsin _0ial Primate Research Center. A preliminary report of this work has WPfwai in Biology of Reproduction 34 (SuppL l):186 (1986). t Rspmu requests and present address: Patricia A. Franks. University of Campus, Department of Psychology. Mississauga, Ontario, 1983), Reproductive anomalies range from sterility, menstrual disturbances, premature rupture of the amniotic membrane, premature delivery, and decreased birth weight to chromosomal aberrations,, macrocephaly, mis carriage, stillbirths, and early death of offspring (Fomi et al., 1980; Nordstrom et al., 1979; Fahim et al., 1976; Banova, 1972). Results from controlled animal studies indicate that lead has an equally wide-ranging and profound effect on several aspects of reproductive function as well as on embryonic development. For example, studies have linked oral lead exposure to delayed vaginal opening in immature female rats (Kimmel et al,, 1980), Adult female mice and rats exhibited irregularity of estrous cycles and anestrus when given lead by various routes (oral: Hilderbrand et al., 1973; s.c. injection: Lach and Srebro, 1972; s.c. injection: Der et ai,, 1974). Cessation of menses, reduced sex-skin color, and decreased num bers of secondary and tertiary follicles were observed in female rhesus monkeys injected with doses of lead that produced overt toxicity (Vermande-Van Eck and Meigs, 1960). Female rhesus monkeys exposed to subclinical levels of lead in their drinking water showed 1055 DUP04Q007209 1056 FRANKS ET AL. significantly longer and more variable menstrual cycles as well as reduced frequency of menstrual cycles and shorter duration of menses (Laughlin et al., 1987). Lead poisoning in macaques, induced by administering lead acetate i.m. daily during pregnancy, resulted in abortion (Tacfaon et al., 1983). Similar findings in rodents sug gest that lead acts to reduce the number of offspring bom to rnthe'S rccrving lead by different routes dur ing i recranr/ (oral: Stowe and Goyer. 1971; Schroeder a..d Mit-'bn jr, 1971 Jacquer et al, 1977; Gdenbro and Rihlstiom, lu7"; and i.v.: McClain and Becker, 1975). Seduced reproductive success in mice treat<.d v/iib lead iiv. has been associated with the preimplant:ticn period of enli'ouc cevelopmcn* ('vVide and Nilsson, 1977). Spec lie effects of lead on the ovary have been repojtu in rati and monkeys (Vermands-Van Eck and Meijs. '960, Stows and Gojcr. 1971; Hildcrbrand ct al., 1913. In menkeys, the Jesuits were flat, v-hitc, atrc-jc z' arias wrh no dorm iant tolhcle:. Fodicular cysts vere loied m ruts. In both species, decreased tolncun dc <elcpment was apj arem. Hawewr. :o date, the ' ffut thit tea,i cxt-ris- on tie ovarrn hi.-micnes has bevn war ou^trated only in pregnant mice. At the time Of blasuicy'-, in.pJ.- nation (Dav 5), examination of hor mone 'i '.c I1: connrmuit with iead exposure 1 howed lover tliUi nornJ progesterone <Pi) Ie.ei' arid degen- eratul corpora lutea By Day 6, estrogen (Hy levels weic- ah. below ccntrol values (Jacque: et al. 1977; "W<ue ni< Nils-ot,, ir'77; Gderbro et al., Admin istration at rd Pi 0D Day 5 reversed tie mnibiiory efPorts of '(.i'd on impitnianon (Wide, 1980). Hav'e^er. the mcrhauism oi action has not been clarified ft has been pept ifcu mar K.a ,nh b ts secrekoii ot reicid horrnf iei acquit, 1977) and/or alters lUionr. of these hoimoic. by reining receptor concentrations (Wide aw; Nil, >g p, Jn7 , '"me urn id-', 198bi Mumaiive- ly it ` ft; Ken svcgef-ifid that lead interferes with ovar ian steroid rccf purs j1 the arem, by inhibiting bmduig and tbu- inte-fenng nith implantation r'oiure s* al,, 1977). The present nudy was undertaken to characterize pctteriis c" circulating levels of P4 in female rhesus mrrl c;u which treatment with lead resulted in aber rant n'erstruai cycles (Laughlin et iL, 1987). In particu lar, w. wished to determine if ovulation was occurring and to assess adequacy of luteal function. MATERIALS AND METHODS Subjects were 17 feral-bom female rhesus monkeys (Macaco, mulatto), 10 experimental and 7 control. The two groups were similar in age, number of previous pregnancies, and parental experience. Hie experimental animals had been treated with lead during 3 periods of approximately * 14 mo each in 1976 -1977, 1978 -1979, and 1980-1981 (in conjunction with breeding experiments), and again for 33 mo in 1982-1984 (in conjunction with behavioral studies). Lead acetate was added to distilled water and treat ments were at approximately the same levels .of lead intake for each of the four exposures (see Ldughlin et al., 1987). We report here on the last 7 mo of the fourth exposure, from April through October 1984. Estimated agi of the subjects at that time was 16-20 yr. Lead was administered to the experimental subjects 5 days/wk. Amounts were adjusted weekly to maintain circulating blood lead levels near a target of 70 jxg/dl. Control levels were < 10 pg/dl Doses required to maintain this level for individual nonkeys ranged from 2 to 8 mg/kg/day with a mean aose of 5.4 mg/kg/day, 5 days/wk. A target 'cvcl of 70 fig/cl was chosen as the highest amount that would resuit in no central nervous syct',m alterations nor produce clinical symptoms such at appetite loss, weight loss, or hematocrit depression. Animals were weighed every 2 wk. Animals were checked 6 davs/v/k for the presence of menstrual blood, and the first day of bleeding war defined as Day 1 of the menstrual cycle. BJood samples were obtained from each monkey twice a mend', for analysis of lead and zinc protopor phyrin (Z.PP). A'l samples were analyzed for ZPP; howeve-. because blood lead values were stable, only 3 samples (selected at approximately equal intervals) from experimental subjects and 1 sample from controls were analyzed for Wood lead levels. Hematocrit vol ume. measured from the same blood samples, was de termined only once. Blood samples for P4 analysis were taken 3 times/wk CM, W. F) between 0800 and 1000 h beginning on Day 10 of die menstrual cycle, to coincide with o'mlaticn and the beginning of the luteal phase. If menstruation had not occurred by Day 30, frequency of sampling was reduced to once a week. Blood iead levels * ere analyzed by atomic absorp tion. spectrophotometry at the Harlow Primate Labora tory or at the Wisconsin State Laboratory of Hygiene in Madisrn, WI ZFP levels were, determined with a hemaiofluororneicr (AVIV, Lakewo o g , NJ), and hematocrit levels were measured by capillary centrifugation at the Harlow Primate Laboratory. Pianna concentration" of P4 were determined by a radioimmunoassay differing from that originally re ported by Clark et al. (1978) in that 1) the antibody DUP040007210 LUTEAL DYSFUNCTION ASSOCIATED WITH Pb EXPOSURE 1057 (Holly Hill Biologicals, Inc., Hillsboro, OR) was raised in rabbits against a conjugate of progesterone-11-succinyl bovine serum albumin and its specificity was verified lo be adequate for unchromatographed samples; 2) 20 j.ti of plasma was assayed; and 3) the range of the standard curve was 4 - 250 pg, with an average 50% inhibition point of 23.8 2.0 pg (mean SD). Parallels \ ism was confirmed between the plasma samples and the standard curve. The average intra- and interassay coef ficients of variation, were 9.8% and 13.8%, respectively; the extraction efficiency was 82.3% 4.0%; and the : average minimal sensitivity of the assay was 2.5 0.4 PS- .. Plasma Pi levels were graphed using am Interactive Digital lor.er to give a profile over time (days) for each animal (Fig. 1 and 2). Relative area under the curve was measured from each profile with a Terak 39510/jl)a Graphic Computer System (Treralc Coip., Scottsd tic, AJZ). % St:ti:trca analysis of differences between experitJ mental and control groups for the measures of blood '% lean and bo< y weight was performed with Student's rtest. Group nean differences in P4 were analyzed by "3~ ANOYA for variables with repeated measured and oth -ff erwivs with Student's r-tesL All results have been preseni^d .is the mean SEM. In addition, individual values fc- plasma (both the area under the curve and ; the max mum heigat) were analyzed against lead dose, b blco-1 leer, level, or ZPP by regression analysis on a .1 - computer program (Cricket Graph, Cricket Software, Hw Inc.. Phi advlpbia, PA). For all statistics, a p value <0.05 was considered to be significant. RESULTS Effects of Lead Exposure on Ceneml Health AH animals maintained stable body weights during the study* and there was no significant difference be tween control (7.08 0.36 kg) and treated (7.33 0.77 kg) females. The mean blood lead level of experimental ahhnals was 68.9 6.54 pg/100 ml; values for file control females were <10 {Xg/100 ml. Concentrations of PP were significantly greater for treated animals (107.4 12.0 pg/dl blood) than for control animals (14.7 14.4 ftg/dl blood). Hematocrit values ranged from 36% to 44% and did not differ significantly between groups. 711616' was no significant correlation between lead intake and blood lead level (r=0.57) or between lead intake and ZPP concentration (r=0.56) among the experimental monkeys. This was not surpris ing given that lead intake was adjusted to maintain equivalent blood lead levels across all the treated mon keys, thus restricting the range of correlation. Relationship between Menstrual Cycles and Plasma Progesterone Patterns for Individual Subjects Menstrual cycles were considered anovulatory when menstruatirin was preceded by a maximum amount of P4 lower than 1.0 ng/ml. There was no significant difference between control and experimental subjects in the mean percent of anovulatory cycles (31% 8.7 vs. 40% 12.3, respectively). Time-line graphs are shown for females which exem plify the most regular, average, and least regular pat terns of P4 profiles for control (Fig. 1) and experimental (Fig. 2) groups. Figure la shows the P4 curve for the control subject (Con 7 in Table 1) with the most regular pattern; Con 5 in Table 1 shows a similar curve, indicating regular and ovulatory cycles for both of these control monkeys across aH 7 mo of the study. A control animal (Con 4) that had ovulatory cycles in spring and fall, but not in summer, is shown in Figure lb; Con 2 also exhibited this, pattern. The curve for 1 of the 3 control animals with the lowest or least frequent P4 peaks is shown in Figure lc (Con 1). The monkey represented in Figure lc showed ovulatory cycles in the spring but none in summer, and it did not resume ovulation by the end of the study in fall. Con 6 exhib ited this overall pattern, having only one P4 peak, of about 3 ng/ml, in the spring. Con 3 had a pattern similar to that of Con 1, except for the occurrence of an additional ovulatory cycle in fall. In contrast to the control subjects, only one experi mental animal, Pb6, showed a pattern of regular ovula tory cycles similar to that of the most regular control subject represented in Figure la. Nevertheless, P4 peaks for this monkey were lower than for the most regular controls. Three additional experimental monkeys (Pb8, Pb9, and PblQ) showed ovulatory cycles in both spring and fall but not in summer (Fig. 2a). P4 peaks for these 3 monkeys also were markedly lower than for the con trols in Figure 1, a and b. Five experimental monkeys (Pbl, Pb2, Pb3, Pb4, and Pb7) had curves similar to the curve for Pb7 (shown in Fig. 2b), a monkey that had neither the most regular nor DUP04D007211 1058 Panel a FRANKS ET AL. Panel b Panel c Panel c FIG. 1. Progesterone profiles to control subjects forth? 7 mo of the study. Panela: anexsinple ofmostregular paitenu/'onei b: anexample ofanaycragc pattern: Panel c: an example ofleast regular pattern. Solidblocks correspond to -observed menstrual bleeding. spond to observed menstrual bleeding. DUP040007212 LUTEAL DYSFUNCTION ASSOCIATED WITH Pb EXPOSURE TABLE 1. Summary' ofdata pn individual subjects, shown as means and standard errors ofrepeated measures per subject. Subject Body weiglit (kg) (n = 14)a Pb dose (mg/kg/day) Total #of PbB ZPP menstrual periods; (pg/dl blood) ([ig/dl blood) P4 peaks (), & (n = 3)s (n = I5)a ovulatory P4 peaks Overall P4 peak heights, ng/ml; and mean Srea/Day*' Spring Summer Fall Pb-1 Fb-2 Pb-3 v- .*! fM '?. Pb-5 * ' Pb-6 M -. Pb-7 Pb-8 '$ty Pb-9 pwo rj3 Con-1 tJon-2 JglreL, Con-3 ;S_. Con-4 fEtr * ir' Con-5 . Con-6 3|E7" - , Con-7 5.59 0.33 6.80 fl#8' 9.10 0.44 7.12 0.38 7.77 Q.18 6.02 0.32 8.42 fit 5.86 0.23 iSf 0*2 J.34 0.73 0.11 021 0.31 0.40 111 0.15 4.46 0.21 6.22 til' 5.05 . Sl.36 c!|| 5.0 6.0 7.0 5.0 8.0 3.0 7.0 4.0 2.0 7.0 NA NA NA NS NA NA NA 593 11.9 963 313 68.3 19.1 43.7 9.3 43.7 203 94.0 59.7 58.0 16.4 603 15.5 99.0 42.8 663 25.0 4.(f (1/5/84) 1.0 (2/7/85) 4.0 C7/5/84) 3.0 (10/4/84) 1.0 (10/17/85) 3.0 (4/12/84) 3.0 <10/6/83) 69.6 15.49 99.9 1032 187.6 16.80 114.9 20.07 1483 17.95 92.9 17.72 118.2 16.88 56.0 832 96.0 20.93 89.9 18.70 13.9 2.29 14.9 2.i; 16.8 2.93 12.3 2.99 83 233 20.6 3.42 16.0 S.&9 4 Cl). 1 6 ,(2). 1 2 a). 2 4 (2). 2 2 (0).0 5 (6). 6 .4 m. 1 6 (7). 6 .6 (6), 6 7 m. 4 5 (5); 3 6 (3). 3 5 (3), 3 7 (S).5 8 0,7 4 (1). 1 9 (8). 8 *n = Minimum number of scores averaged per! subject, with' noican (upper) and SD (lower). 0.49 (12.2) 0.61 (It)) *- 0.73 (203) 2.69 (39.4) 032 (7.4) 1.43 (38.1) 1.60 (253) 1.60 (27.4) 1,11 (23.1) 1.49 (373) -- 330 (65.8) 2.81 (60.0) - 3.68 (54.4) 137 (313) 133 (283) 132 (25.7) 1.75 (483) _ 1.88 (373) 057 (22.1) 1.40 (413) 1.72 (44.4) 1.07 (24.0) 1.84 (510) 3,12 (88;6) 1.48 (25,2) 3.73 (1263) 2 83 (523) 2 95 (553) 4:75 (74.7) 0.12 (1-3) 031 (53) 0.63 (0.0) 0.16 (4.9) 0.05 (03) 3.56 (66.4) 0.00 (0.0) 0.88 (2017) 1.64 (2.1) 0:67 (46.7) .067 (133) 1.21 (22.8) 0.37 (4.24) 234 (343) 2.09 (433) 0.10 (2A) 231 (49.0) 0.09 (4.0) 0.09 (2-8) s 038 (7.6) * 2.62 (14.4) 0.00 (0.0) 300 (523) 1.44 (29.4) 3.06 (40.8) 0,81 (5.1) 0.15 (0,6) " 4.44 (37.8) 3.52 (84,6) 3.78 (39.6) 'Tor each subject, mean P4 peak height is shown on die fust Uric as an open number, and mean P4 area is shown on.the second line in parentheses: data are based bn 3/wk sampling. AH numbers are mean/season/subject. cOne bipod sample was assayed per.control subject, taken on the date jnparentheses. All experimental blood lead values were rfe.n-rm^pH from blood saiimled between 4/1/84 iitf 11/2/84. K dNo 3/wk sampling in seasons in which the subject failed to shew mehstnial bleeding. ^ast regular pattern of P4 profiles of these 5. Note the small ovulatory peak in spring, followed by an ano vulatory cycle, with no further ovulatory cycles during the summer and fall months. This subject had no detect able levels of P4. during the summer and fail. The other 4 monkeys showed small increases of P4 in summer and i although none of the increases exceeded 0.3 ng/fnl. ne monkey (Fb3) had one ovulatory cycle in late spring and .mother in early summer. A second monkey (Pbl) had only one ovulatory peak, and that occurred in Spring. A third monkey (Pb2) had one ovulatory cycle tft spring and one in summer. The fourth monkey (Pb4) ad 311 ovulatory and an anovulatory cycle in spring and 3 Srnall anovulatory peak in summer; die two anovula- C;- ^ Peaks were not associated with menstrual bleeding. Finally, one experimental monkey (Pb5) exhibited the curve shown in Figure 2c, This monkey had very little P4 secretion and only two periods of menstrual bleeding during the entire 7 mo of the study, both occurring in the summer months. It is pertinent that this monkey received the highest lead dose of all (8 mg/kg/ day) and had the second highest mean ZPP level, yet had the lowest mean blood lead level in the group. By contrast, the animal receiving die lowest lead dose of all (Pb9 at 2 mg/kg/day) had a mean ZPP level in the midrange and the highest mean blood lead level; she had a P4 pattern virtually identical to that in Figure 2a, with low peaks aid with six ovulatory cycles over the 7 mo of the study. In addition, die experimental monkey with the most regular cycles (not shown) also received DUP040007213 1060 FRANKS ET AL. TABLE 2. .Effects oflead on progesterone measures. Group Control Lead IT 7 10 Area under curve {relative units)* 6.82 (1.11) 4.07* (0.74) Peak height (ng/ml)* 2.81 (0.31) 1.79 (0.24) Days above Ing/ml 10.54 (1.44) 6.06 (0.635) "Significantly different from controls by 2-tailed t-test; jxQ.05. bSignific amly different from controls by 2-tailed r-tesc p<0.01. Numbers are means {tapper) and SEM (in parentheses). one of the lowest doses of lead (3 mg/kg/day) and had a ZPP and blood lead level in the midrange. Plasma Progesterone Levels Four different characteristics of P4 were analyzed to assess differences between control and experimental monkeys; 1) number of P4 peaks (a peak was deter mined to be a rise in P4 levels above 1 ng/ml as determined from the P4 profile of each animal); 2) height of P4 peaks; 3) area under the curve; and 4) number of days when P4 was above 1 ng/ml. There were no significant differences in actual numbers of P4 peaks between the two groups. However, in other char acteristics, clear differences between groups were not ed. Table 2 compares P4 levels of treated and control animals. Aaimais exposed to lead had only 60% of die P4 values of control animals, which was manifested in a significantly smaller area trader the curve (t=2.16, j s k O.05) and significantly shorter peak heights of P4 (t=2.63, p<0.01). Monkeys exposed to lead also had significantly fewer days per cycle when P4 levels were 1 ng/ml (t=3.10, p<0.01). For all animals in both groups, the frequency of blood sampling was 3/wk starting on Day 10 of the cycle, but was reduced to 1/wk following Day 30 of the cycle if no menstruation occurred in that interval (see Materials and Methods), To ensure that the method of sampling did not affect these analyses, the P4 data were analyzed further using Only those calendar periods for each subject in which blood samples were taken 3 times/wk. '[his assured exactly the same sampling den sity for both the experimental and control groups. The P4 curves during only these more frequent sampling periods were then analyzed by measuring each subject's P4 peak height (ng/ml) and mean P4 area (digitizer units per day); P4 area Was expressed per day to compensate for variation in the durations of the 3-wk sampling periods. The regression analyses for lead intake, blood lead levels, and ZPP levels are shown by season in Table 3. From the regression analyses (Table 3), it can be seen that the P4 areas declined significantly with in creasing lead intake, increasing blood lead levels, and increasing levels of ZPP. The P4 peak heights were more variable. They declined significantly wifir increas ing lead intake and were not significantly correlated with blood lead level. The correlation between peak height and ZPP level approached significance (p<0.10). In summary, these regressions demonstrated that, higher lead intake was associated with reduced Pa areas and lower P4 heights. Furthermore, area and height each correlated about equally with lead intake, but area correlated more strongly than did height when regressed against blood lead level and ZPP. 1 he seasonal effect on P4 peak height was tested by ANOVA with lead treatment as a between-subjects vrliable fiead vs. control) and season as a withinsubjrct variable (3 seasons). Four subjects failed to exhibit menstrual bleeding within some season, thereby precluding a period of 3/wk sampling during that sea son. Each subject had missing points in fall, and one treated subject also had a missing point in summer. Since 2 subjects were experimental and 2 were control, suggesting no undue treatment bias, those subjects with missing data points were eliminated from the ANOVA. This avoided the uncertainty of estimating missing data points. Tims, the ANOVA was run on the 13 subjects for which overall P4 means are shown in Table 1. The ANOVA on P4 height shows that the main effect of lead treatment was significant (F=7.10, df=7,ll; p=0.022), the seasonal effect was significant (F=6.70, df=2,22; /7=O.QQ5), and the interaction was nonsignifi cant (F=l.bl. df=2,22; p=0.22). Analysis on the mea sure of P4 area gave similar results. Thus these results gave no support for treating the effect of lead as differential across the three seasons. In summary, the experimental monkeys had reduced secretion of P4 as shown by lower P4 area under the curve over the 7 mo of the study. P4 secretion varied by season, but not at differential rates for the two groups. DISCUSSION In this study, chronic exposure to lead clearly had a detrimental impact oh the luteal phase of ovarian func- DUP040007214 LUTEAL DYSFUNCTION ASSOCIATED WITH Pb EXPOSURE 1061 X/U3LE3. Linearregressions on leadintake, onblood lead level,and on ZPPfor the two progesterone measures,maximum peak height, and total area, as quantified for only those time periods at which blood wassampled 3 times/wk. Lead intake and related measures; (a = 13 subjects; sec text) jjsd intake (ragkgfaay) pbB (tt&'dl) Zpp ([tg/dl blood) intercept 46.7 45.5 52.1 Peak area (digitizer unns/day) Slope T -4.46 -021 -0.30 -.61* -56b .-,70s intercept 2.51 izr 2-40 Maximum peak height (ng/ml) Slope r -0.27 -0.01 -0.01 -.63* -.43 -51c jxom. &p<0.05. <7X0.10. tion. Significant differences were found between con trol and experimental subjects in three of the four measures of P4 secretion used to evaluate luteal func tion: area under the curve, peak heights of P4, and mean number of days per ovulatory Cycle when P4 was above 1 ng/ml. In all three of these measures, females exposed to lead showed significantly reduced amounts of P4 secreted by the ovary. The fourth measure of progester one activity, total number of P4 peaks, as well as number of Eitovulatory cycles, did not yield differences between control and lead-treated animals. Although blood lead and ZPP levels were elevated during treat ment, the monkeys exhibited no overt signs of toxicity. Hematocrit values and body weights of the treated animals did not differ from control animals. Thus, these effects of lead on luteal function occurred in the ab sence of overt effects on the general health of the experimental females. Although there were seasonal changes in P4 activity, as is typical of monkeys housed in laboratory environ ments (Hut;: et al., 1985; Daily and Neill, 1981), the patterns of change were similar for both control and treated monkeys. That is, although peak height and area of P4 changed across the seasons, die magnitude of the change was not different for the two groups of mon keys. In the previous study, probability of cycles of normal length also declined during summer and fall compared to spring, but not differentially for experi mental and control monkeys (Laughlin et al., 1987), Further, there was substantial cycle activity during the months of increased cycle length, even for the treated monkeys. These effects On ovarian cycle extend findings from previous study with the same animals in which ^cad-treated monkeys woe found to have fewer cycles, 0nger and more variable intercycle intervals, and fewer ^ys of vaginal bleeding per cycle than control mon keys (Laughlin et al., 1987), Three of these four alter ations became apparent only after lead exposure had been continued for many years, while the fourth, dura tion of vaginal bleeding, was disrupted by lead during the initial year of intake. All four effects of lead on menstrual cycles remained apparent for at least 1 yr after lead intake was discontinued foEowing a total of approximately 6 yr of lead administration. The longer and more variable intercycle intervals observed in the previous study suggest that lead has effects on the foEicular stage of the cycle, since the postovulatory period is relatively constant regardless of total cycle length (Reid et al., 1972). Results of the present study, obtained during the last 7 mo of lead intake, extend these effects of lead to include luteal function. Al though menstruation and progesterone activity do not cease in response to prolonged chronic exposure to lead, both are clearly attenuated, which could result in reduced fertility. Whether the alteration in luteal function demon strated in the present study was due to direct effects of lead upon the ovary or to changes in the hypothalamicpituitary axis is not clear. Some studies suggest the possibility of direct effects of lead on ovaries. In rhesus monkeys, treatment with lead resulted in reduced num bers of secondary and tertiary fofiicies and increased follicular atresia (Vermande-Van Eck and Meigs, 1960). Ovarian weight (Der et al., 1974) and number of corpora lutea (Hilderbrand, et al., 1974) were reduced in rats treated with lead. Also, fish exposed to low levels of lead showed reduced ovarian weight (Katti and Sathyanesan, 1983; Sastry mid Agrawal, 1979). Alternatively, other reports suggest that lead may act at the level of the hypothalamus anti pituitary. For example, sexual maturation, thought to be mediated by the hypothalamus (Firfit and Schwartz, 1977), was de layed in female rodents exposed chronically to low lead D U P040007215 1062 FRANKS ET AL. levels (Der et al., 1974; Grant et al., 1980; Kimmel et aL, 1980). It has been demonstrated that high levels of lead result in abolition of the surge of follicle-stimulat ing hormone (FSH) in prepubertal rats (Petrusz et al., 1979). This surge normally occurs at 15 days of age (Kragl et al., 1972; Ramirez, 1973; Uilenbrock et al., 1976; Kronibus and Wuttke, 1977) and is required for normal puberty. The possibility that lead interferes with FSH urges suggests that suppression of this increase during the Follicular phase of the menstrual cycle in the present stniy could have been responsible for the defi cient uteal function noted (Nass et al., 1979). Thus, although our demonstration of decreased P4 levels,'concomirmt with alterations in menstrual cycles clearly ;mi: lie 5 o'-.triur dysfunction, multiple loci within the hyooi t alpituita-y-ovarian axis may be mediating this action of lead. Tiiix study points out die subtlety with which lead can ac:t. Exposure to lead at levels that do not cause overt loxicity can nevertheless exert demonstrably neg ative effects upon ovarian function. ACKNOWLEDGMENTS We wish to acknowledge and thank those who devoted extensive tune to collecting blood for this project: Mark E. Cupp, Pamela Ellington, Barry K, Hartnp, Scott Laska, and Boni Westover. We also wish to thank Or. 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