Estradiol-induced follicular atresia in rhesus monkeys is not prevented by exogenous gonadotropins.код для вставкиСкачать
American Journal of Primatology 23:247-255 (1991) BRIEF REPORTS Estradiol-I nduced FolIicuIar At resia in Rhesus Monkeys Is Not Prevented by Exogenous Gonadotropins REINHOLD J. HUTZ,' DONALD. J. DIERSCHKE? AND RICHARD C. WOLF2 'Department of Biological Sciences, Uniuersity of Wisconsin, Milwaukee; and 2Wisconsin Regional Primate Research Center, Madison, Wisconsin Estradiol-17P (E,) induces atresia of the dominant preovulatory follicle (DF) when administered on day 6 of the menstrual cycle. The present study was designed to determine whether the atretogenic effect of E2 could be averted by the administration of exogenous gonadotropins, in an attempt to determine whether E,-induced atresia in primates is due to a direct action at the ovarian level or is mediated via pituitary secretion. After identification of the DF during laparoscopy, cyclic monkeys received Silastic capsules containing E, placed S.C.for 24 hours, plus one of the following treatments: phosphate-buffered saline, or 25 I.U. of either human urinary menopausal gonadotropin (hMG), FSH-rich hMG, human urinary FSH (uFSH), or human pituitary FSH (pFSH) injected i.m. twice daily for 2 days. The control treatment resulted in atresia of the DF and extended follicular phases (26.3 5.9 days, x % S.D.), but in normal luteal phases following ovulation of a substitute DF. Similar results occurred in all animals receiving FSH-rich hMG or pFSH, and in 11 of 16 animals receiving hMG or uFSH (P > 0.05). Since all possible routes and regimens of gonadotropin administration were not attempted, a central action of E, cannot be ruled out. However, we believe that the experimental observations support our contention that the atretogenic action of E, is exerted in part a t the ovary. * Key words: Mucuca muluttu, steroids, follicle-stimulating hormone, preovulatory follicle INTRODUCTION We have demonstrated previously that estradiol-17P (E,), administered S.C.in Silastic capsules for 24 hours on day 6 of the menstrual cycle, induced atresia of the dominant preovulatory follicle (DF) [Dierschke et al., 19851; this allowed for the ovulation of a substitute follicle and for a marked extension of the follicular phase. During treatment, circulating amounts of follicle-stimulating hormone (FSH) de- Received for publication August 9, 1990; revision accepted October 18, 1990. Address reprint requests to Dr. Donald J. Dierschke, Wisconsin Regional Primate Research Center, 1223 Capitol Ct., Madison, WI 53715-1299. 0 1991 Wiley-Liss, Inc. 248 I Hutz et al. clined transiently, and showed a rebound after removal of the capsules; the amounts of luteinizing hormone (LH) did not change consistently [Dierschke et al., 19851. Local intrafollicular effects of E,, administered s.c., were confirmed by studying contents of the DF obtained by aspiration on day 10 of the cycle. Viability and steroidogenic capability of granulosa cells were reduced distinctly; the binding of FSH t o granulosa cells was not altered, but FSH added in culture had limited capability to reverse the effect of estrogen [Hutz et al., 19861. E,, administered locally by injection into the stroma of the ovary containing the DF, also induced atresia in the majority of animals; this treatment induced brief increases in circulating amounts of estrogen at six hours after injection in some animals, but did not alter circulating amounts of FSH or LH [Hutz et al., 19881. In related studies with hamsters and monkeys, direct exposure of granulosa cells to E, in long-term culture resulted in their reduced steroidogenic capability [Hutz et al., 1987a, 1987b, 19891. Although much of the data available to date tends to support our hypothesis that estrogen may induce atresia of the DF by an action at the ovarian level, we cannot rule out the possibility that altered secretion of gonadotropins by the pituitary is also a factor [Zeleznik et al., 19853. The present study was done to test this possibility by using gonadotropin preparations having a variety of potencies as replacement therapy, in conjunction with estrogen treatment. METHODS Experimental Animals and Treatments Thirty adult female rhesus monkeys (Macaca rnulatta), exhibiting normal menstrual cycles of 24-32 days in length and estimated luteal phases of > 10 days preceding treatment, were used. The animals weighed 4-8 kg and were 6-20 years of age. Animal care and housing, blood sampling, and surgeries were as previously described [Hutz et al., 19851.The DF was identified during laparoscopy on day 5 or 6 of the cycle. To induce atresia of the DF, 4-9 animals per group were treated with 4 Silastic capsules containing E, placed S.C.for 24 hours from the morning of day 6 (capsule preparation was as described previously [Dierschke et al., 19851). Other treatments superimposed on the above were: phosphate-buffered saline (PBS, control); human urinary menopausal gonadotropin (hMG, Pergonal; FSH:LH = 1:l; Serono, Randolph, MA), FSH-rich hMG (Neo-pergonal; FSH:LH = 3:l; Searle Laboratories, Montrouge, France); human urinary FSH (uFSH; > 99% FSH activity; Metrodin, Serono, Randolph, MA); and human pituitary FSH (pFSH). PBS or gonadotropin was administered i.m. in doses of 25 I.U. twice daily for 2 days (total dosage, 100 I.U.), starting when the estrogen implants were installed. Blood samples were obtained daily, or more frequently (at 0800, 1500, and 2100 h) during treatment days. Additional observations by laparoscopy were done at about day 15 of the cycle to evaluate ovulation or atresia of the original DF and were also done later if necessary to evaluate ovulation of a substitute follicle [Dierschke et al., 19851. In a preliminary experiment, animals were treated with the atretogenic regimen of estrogen described above, and either hMG or uFSH was administered as replacement therapy at a lower dosage (7.5 I.U.) but for a longer period (twice daily for 4 days; total dosage, 60 I.U.). Normal ovulations of the original DF occurred in 0 of 5 controls, 2 of 7 treated with hMG, and 3 of 9 treated with uFSH. On the basis of these preliminary observations, we selected the higher dosage for the present study and concentrated the replacement therapy during the interval when endogenous FSH tended to be suppressed by estrogen treatment. Gonadotropin and Estrogen on Monkey Follicles / 249 TABLE I. Effect of Exogenous Gonadotropins on Ovulation and Cycle Length in Rhesus Monkeys Treated With Estradiol-17P No. of animals in which atresia occurred/total' Cycle length/length of luteal phase, days' Saline vehicle 515 40.3 i 4.6 (4)" 23 (1) 114.0 k 1.4 (4)" /anovulatory (1) hMG 5/7 23 (2) 25.3 i 2.9 (3)' 51.0 t 15.6 113.0 t 2.8 (2)" lanovulatory (3) 114.0 i 4.2 (2)" FSH-rich hMG 515 43.7 uFSH 6/9 4.6 (3)" 33 (1) 24.7 t 2.1 (3)' 21.5 t 6.4 (2)bc 36 (3) 114.0 2 2.0 (3)" lanovulatory (2) /17.0 t 2.0 (3)" /anovulatory ( 2 ) /15.0 -+ 1.4 (2Ia pFSH 414 33.0 i 4.1 (4Ib /13.3 i 1.9 (4)" ? 'There were no significant differences among treatment groups by the Fisher exact-probabilitytest (P > 0.05). 2Valuesare x 2 1 S.D. Numbers in parentheses denote observations.Cycle lengths of approximately 24 days and ail luteal phases are in the normal range. Some animals were deleted because of incomplete cycle information. FSH-richhMG and uFSH-treatedmonkeys in which ovulation occurred were further divided into 2 groups based upon statistically different cycle lengths. a,b%uperscriptswithin a column that do not contain a common letter indicate significant differences ( P < 0.05). Radioimmunoassays Serum concentrations of LH, FSH, estrogen (El, and progesterone (P) were determined by methods described previously [Clark et al., 1978, 19791. Serial dilutions of the various FSH preparations exhibited linearity in the FSH assay over the range 1:0 to 1:lOOO. PFSH cross-reacted 100% in our assay. The LH present in hMG and FSH-rich hMG did not cross-react perceptibly in the LH assay. Statistical analyses Fisher's exact test [Zar, 19741 was used to compare ratios in Table I after pooling similar data. Lengths of cycle and luteal phase were analyzed by completely randomized one-way analysis of variance (ANOVA) [Zar, 19741followed by Student-Newman-Keuls (SNK) test (Table I). Amounts of circulating hormones were quantitated by integrating and digitizing the areas under hormone curves [Dierschke et al., 19851, and analyzed by one-way ANOVA and SNK (Table 11). RESULTS In rhesus monkeys, prevention of estrogen-induced atresia of the DF and concomitant extension of the follicular phase did not occur to a significant extent, regardless of the superimposition of four different gonadotropin preparations possessing from 50 to 100% FSH activity (Table I). Examples of a control animal in which atresia of the DF resulted and a uFSH-treated animal in which ovulation occurred are shown in Figures 1 and 2. Atresia was induced in 25 of 30 animals, including all animals receiving saline, FSH-rich hMG, and pFSH; ovulations occurred only in 2 of those receiving hMG and 3 receiving uFSH (Table I) (P > 0.05). All luteal phases, whether derived from the original DF or from the substitute follicle, were of normal duration (Table I). Total circulating amounts of LH, as measured by digitized areas under hormone curves, were not consistently affected, regardless of whether cycles were 250 / Hutz et al. TABLE 11. Effect of Exogenous Gonadotropins on Hormone Patterns in Rhesus Monkeys Treated with Estradiol-l7P, Total Area Under Hormone Curves* FSH Estrogen Proge s te rone Saline vehicle 0.86 f 0.08 (4)' 0.81 LH (atresia) (anov.) 0.97 t 0.04 (4)" 1.03 0.74 ? 0.02 (4)" 0.64 0.60 2 0.09 (4)"' 0.31 hMG 0.34 2 0.10 (2Iab 0.32 ? 0.01 (2)" 0.57 t 0.25 (3)"'' (ovul.) (atresia) (anov.) 1.02 ? 0.11 (2)" 0.89 ? 0.02 (2p 1.20 t 0.30 (3)" 1.34 2 0.08 (2)'' 0.81 0.09 (2Ib 0.56 2 0.03 (2Iab 0.89 0.73 ? 0.15 (3)" 0.34 t 0.09 (3)" FSH-rich hMG 0.37 i 0.03 (3Yb 0.36 2 0.12 (2)" (atresia) (anov.) 0.95 ? 0.03 (3)" 0.91 t 0.04 (2)" 0.78 2 0.08 (3)" 0.57 t 0.01 (3Iab 0.57 ? 0.20 (2Iab 0.52 2 0.32 (2)ab uFSH 0.71 t 0.12 (3)bc (ovul.) 0.44 ? 0.07 (3)" (atresia) (anov.) 0.26 1.28 t 0.16 (3)" 1.35 ? 0.24 (3)" 0.91 1.15 f 0.05 (3)'' 0.90 f 0.09 (3)"' 0.80 0.77 2 0.20 (3Fb 0.71 t 0.19 (3Fb 0.06 hFSH 0.62 t 0.07 (3Iab' 0.25 1.18 t 0.14 (3) 1.59 0.85 2 0.03 (3)" 0.62 0.53 2 0.16 (3)"' 0.54 (atresia) (anov.) * *Relative units ( 2 1 SEM) for integrated areas under curves using X-axis a s the base; normalized per day. Numbers within parentheses denote observations. Values for LH and FSH, E, and P represent the entire cycle, follicular phase, and luteal phase, respectively. ovul. = ovulatory cycle. atresia = cycle in which atresia of dominant follicle resulted. anov. = anovulatory cycle. ",bscSuperscriptswithin a column that do not contain a common letter indicate significant differences (P< 0.05). ovulatory or anovulatory, or whether or not atresia of the original DF resulted, and regardless of the type of exogenous gonadotropin therapy used (Table 11). FSH concentrations were likewise not significantly altered with uFSH administration (Table 11). Total estrogen and progesterone were expectedly elevated in ovulatory vs. anovulatory cycles (Table 11). DISCUSSION The present experiment indicated that the atretogenic action of estradiol-17P on the DF of rhesus monkeys was an all-or-none event, as it was not forestalled to any appreciable extent, regardless of the type or dosage of FSH preparation superimposed upon treatment. FSH-rich hMG and pFSH had no effect, while hMG and uFSH prevented E,-induced atresia in a non-significant number of individual animals. Other workers have demonstrated that in rhesus monkeys FSH-rich hMG appeared to compensate partially for the induction of luteal phase defects and FSH suppression by porcine follicular fluid [diZerega & Hodgen, 19811. Our results differ substantially from those of Archer et al. in women, where hMG reversed the atretogenic effect of oral administration of ethinyl estradiol [Archer et al., 19881; the discrepancy between our studies may be attributed to a number of factors, among them species differences, route, composition and metabolism of administered agents, and length of treatment. Exogenous FSH preparations appeared, in some instances, to mask or mute the "inhibition" and "rebound" phenomena characterizing endogenous FSH after S.C. E, administration [Dierschke et al., 19851. Figure 2 shows that FSH concentrations in a monkey given Metrodin remained at a fairly steady state or were even slightly elevated during treatment with respect to baseline concentrations that existed prior to E, exposure. This elevation was evident across animals, but was not correlated with increased incidence of ovulation. The effect was partly due to the cross-reaction of these preparations in our FSH assay; the reduction in the Gonadotropin and Estrogen on Monkey Follicles / 251 600 550 500 450 I -5 c 400 350 300 250 Y 3 200 - 200 - 100 - 50 150 100 - 50 1 1 . 0 I I I . 1 . I I I . I . I I I I I . I . l . I . I . I . I . I . l 1 1 . I 0 487.3 300 250 200 150 100 50 0 0 2 4 6 tttt 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Day of Cycle Fig. 1. Hormonal patterns in a rhesus monkey treated with four capsules of estradiol-17p (E,) for 24 hours on day 6 of the menstrual cycle (descendingarrow) and with saline vehicle twice daily for 2 days (ascendingarrows). In this animal, treatment with E, resulted in atresia of the original dominant follicle, and extension of the follicular phase with ovulation of a substitute follicle. Treatment resulted in small, transient declines in LH and FSH. After capsule removal, both LH and FSH “rebounded” to large peaks, but only transiently. This hormonal pattern was also typical of monkeys treated with exogenous gonadotropins, but where atresia occurred nevertheless. 252 I Hutz et al. 450 I- RhQ72 - 900 400 - 800 350 - 700 cn - 600 5 - 3 500 - 400 - 300 - - cc) \ 3 Y I I I 200 100 0 I f -&I P 300 - 250 - 200 - 150 - 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 itit Day of Cycle Fig. 2. Hormonal patterns in a rhesus monkey treated with 4 capsules of estradiol-17p (E,) for 24 hours on day 6 of the menstrual cycle (descending arrow) and with 25 I.U. uFSH twice daily for 2 days (ascending arrows). UFSH reversed the atretogenic effect of E, in this animal and allowed for timely ovulation of the original dominant follicle. Treatment resulted i n timely surges of gonadotropins and attenuated any rebound of these hormones following capsule removal. typically exaggerated "rebound" response to E2 withdrawal may also have signaled an involvement of FSH in E,-induced atresia. Interestingly, Figure 2 shows no surge of FSH coincident with that of LH; a possible hypothesis is that the injected FSH enhanced inhibin production, which in turn served to inhibit peripheral FSH [Rivier et al., 19861. Gonadotropin and Estrogen on Monkey Follicles / 253 High-potency, “pure” FSH preparations may advance folliculogenesis [Schenken et al., 19841,and we observed cycle lengths with uFSH (24.7 2.1 ,x S.D. [nl) near the limit of that which we consider to be normal in untreated animals (28.9 -1- 4.4 I2431 [Hutz et al., 19851. Administered in the early follicular phase, prior to day 8 and establishment of follicle dominance, hMG caused a supernormal number of follicles to develop in the cynomolgus macaque [diZerega & Hodgen, 19801. However, at the dosage and duration employed in the present study and in other similar studies [Schenken and Hodgen, 19831, no hyperstimulation was observed with either hMG or uFSH. The LH content or LH-like activity in some preparations has been shown in other studies (e.g., using hCG) to exert anti-folliculogenic effects when administered in the mid to late luteal phase and delay the next LH surge [Goodman and Hodgen, 19821. The presence of LH in our preparations may additionally have altered the cycle by hastening DF atresia [Goodman & Hodgen, 19821, and the biologic half-life of urinary gonadotropins like hMG, uFSH, and pregnant mare’s serum gonadotropin is relatively long compared with that of pituitary preparations [Chappel et al., 1983; Moor et al., 19851. Therefore, another important consideration in understanding the effects of some preparations is the biologic activity of the preparations. Differences in biologic activity can cause disparities between immuno- and bioassayable concentrations of gonadotropins [Schenken et al., 19851. Several workers have demonstrated that, based upon isoelectric points, multiple forms of FSH exist in both pituitary and serum due to differences in sialic acid content [Wide, 1982; Chappel et al., 19831. Increased sialic acid content of urinary gonadotropins enhanced biologic half-life and biologic activity in vivo, although receptorbinding activity was diminished [Chappel et al., 19831; this may account for the few reversals of E,-induced effects by hMG and uFSH. Interestingly, as duration of E, exposure increases, endogenous pituitary FSH forms appear with higher isoelectric points, augmented receptor binding, and reduced sialic acid incorporation [Chappel et al., 1984; Harlin et al., 19861, thereby possessing a reduced biologic half-life; this suggests one locus for a centrally mediated action of E, in the induction of atresia. In conclusion, exogenous gonadotropins exert no significant effect in negating the atretogenic action of E, on rhesus monkey DF. Since only two doses and two durations of several gonadotropin preparations, and only one mode of administration were tested, the present study does not preclude central mediation of E2. However, we believe that the present experiment, taken together with our evidence discussed in the introduction to this report, strongly implicates a role for the atretogenic action of E2 directly at the ovarian level. * * CONCLUSIONS 1. Administration of various gonadotropin preparations to cycling rhesus monkeys did not reliably overcome the atretogenic effect of estradiol on the dominant follicle. 2. The present study, taken together with previous data, strongly implicates the ovary as one site for estradiol’s effect, in addition to a possible central locus. ACKNOWLEDGMENTS We wish to thank Gail Krueger and Pat Meller for performing the hormone assays; Drs. Gary Hodgen, Jones Institute, Norfolk, VA, and Salvatore Raiti, National Hormone and Pituitary Program, Baltimore, MD, for generously supplying 254 / Hutz et al. the FSH-rich hMG and pFSH, respectively; Dr. J im Hutchison and Serono, Randolph, MA for the uFSH; and Alnita Allen and Dorothy Perry for the expert preparation of this manuscript. This study was conducted in accordance with the “Guide for the Care and Use of Laboratory Animals,” NIH Publication No. 85-23 and Public Law 89-544, “The Animal Welfare Act,” August 24, 1966, and its amendments. 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