SHIGERU KANEKO, NOZOMI SATO, KATSUO SATO, and INORU HASHIMOTO

Transcription

1 BIOLOGY OF REPRODUCTION 34, (1986) Changes in Plasma Progesterone, Estradiol, Follicle-Stimulating Hormone and Luteinizing Hormone during Diestrus and Ovulation in Rats with 5-Day Estrous Cycles: Effect of Antibody against Progesterone SHIGERU KANEKO, NOZOMI SATO, KATSUO SATO, and INORU HASHIMOTO Laboratory of Veterinary Physiology, Kitasato University School of Veterinary Medicine and Animal Sciences Towada-shi, Aomori 034, Japan ABSTRACT Progesterone secretion remained significantly higher during diestrus in the 5-day cyclic rat than in the 4-day cyclic animal. Injection of a sufficient amount of antiprogesterone serum (APS) at 2300 h on metestrus in a 5- day cycle advances ovulation and completion of the cycle by I day in the majority of animals (75 and 80%, respectively). Progesterone (250 jig) administered with APS eliminated the effect of the antiserum. Within 2 h after administration of APS, levels of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) elevated significantly, while a significant elevation of plasma estradiol above the control value followed as late as 36 h after the treatment. None of the 5-day cyclic rats treated with APS showed ovulatory increases of FSH and LH at 1700 h on the second day of diestrus, although 3 of the 4 animals receiving the same treatment ovulated by 1100 h on the following day. The onset of ovulatory release of gonadotropins might have been delayed for several hours in these animals. These results indicate that recurrence of the 5-day cycle is due to an elevated progesterone secretion on the morning of diestrus, and suggest that a prolongation of luteal progesterone secretion in an estrous cycle suppresses gonadotropin secretion. Rather than directly blocking the estrogen triggering of ovulatory LH surge, the prolonged secretion of luteal progesterone may delay the estrogen secretion itself, which decreases the threshold of the neural and/or hypophyseal structures for ovulatory LH release. duration of luteal progesterone secretion between the INTRODUCTION It has been observed previously that progesterone secretion remains higher during diestrus (D) in the 5-day cyclic rat than in the 4-day cyclic animal (Roser and Block, 1969; Furudate et al., 1975; van der Schoot and de Greef, 1976; Nequin et al., 1979). Extensive and detailed characterizations of hormonal and morphological events during 4- and 5-day estrous cycles have been made, and three different hypotheses concerning the etiology of the two cycle types have been examined by Nequin et al. (1979). They have suggested that the 5-day cycle is due to the prolonged progesterone secretion from a new crop of corpora lutea during Day D. The present investigation was undertaken to examine this possibility and to discuss causative sequences of the difference in the rats with two cycle lengths. Animals MATERIALS AND METHODS Adult female Holtzman rats ( g) inbred in this laboratory were kept in a temperature-controlled room (22#{176} C) that was artificially illuminated daily from 0500 to 1900 h. They were fed rat chow (CA-i, Clea Japan, Inc., Tokyo, Japan) and water ad libitum. Vaginal smears were examined every morning, and only those rats which had shown more than 3 consecutive regular 4- or 5-day cycles were selected for the present investigation. The 5-day cyclic rat in this colony showed a nucleated vaginal smear on the second day of diestrus (D II) and vaginal cornification on both proestrus and estrus. Administration of A ntiprogesterone Serum (APS) Accepted October 1, Received May 23, Reprint requests. Antiprogesterone serum was generated in 3 male Japanese white rabbits using progesterone-i 1-hemi- 488

2 DIESTROUS PROGESTERONE IN RAT ESTROUS CYCLES 489 succinyl bovine serum albumin (Steraloids, Inc., Wilton, NH) as the antigen. Titer of APS was determined by the method previously described (Abraham, 1974). The amounts of APS that bound 1.0 jig of progesterone in the radioimmunoassay system were calculated on the basis of their titers and designated as 1 unit. The values for APS obtained from the 3 individual rabbits were 1.0, 2.0 and 4.4 ml, respectively. The specificity of each APS was tested by crossreaction studies (Abraham, 1974). Antiprogesterone serum had less than 8.33% cross-reactivity with pregnenolone, androst-4-ene-3, 17-dione, or testosterone. The cross-reactivities of APS with corticosterone and 20a-hydroxypregn-4-en-3-one were 0.86 and 2.0%, respectively, while the value with 5j3-pregnanediol, cortisone, dehydroepiandrosterone, estrone, estradiol, or estriol was less than 0.25%. Prior to use, APS and normal rabbit serum (NRS) were each mixed with charcoal (Norit A, Nakarai Chemicals, Ltd., Kyoto, Japan; 50 mg/ml). Each mixture was stirred at room temperature for 2 h, centrifuged at 10,000 rpm for 30 mm, then filtered through a Mihipore filter (0.45 pm, Japan Millipore Ltd., Tokyo, Japan). The charcoal-treated APS and NRS contained less than 1.0 ng progesterone, 0.25 ng 20a-hydroxypregn-4-en- 3-one and 10 pg estradiol per ml. Each 5-day cyclic rat was given a single intraperitoneal injection of either APS (0.5 or 1.0 unit) or NRS (4.4 ml) at 2300 h on metestrus (M). Progesterone Administration Progesterone (Sigma Chemical Co., St. Louis, MO) was dissolved in sesame oil (250 pg/0.2 ml). Each 5-day cyclic rat was given a single subcutaneous injection of the steroid (250 jig) in combination with APS at 2300 h on Day M. Collection of Systemic Blood Intact 4- and 5-day cyclic rats and 5-day cyclic rats treated with either NRS or APS were killed by decapitation at various times indicated in the Results. Individual blood plasma samples were divided into 3 portions and kept frozen (-80#{176}C) until assayed for each hormone. Radioimmunoassay (RIA) Plasma progesterone and estradiol were extracted by the method previously described (Hashimoto et al., 1975). The residues containing steroids were subjected to Sephadex LH-20 (Pharmacia Fine Chemicals, Piscataway, NJ) column chromatography (6 mm i.d. X 60 mm) using solvent systems of iso-octane/benzene/ methanol (85/15/5, v/v) for separation of progesterone, and benzene/methanol (90/10, v/v) for separation of estradiol. Each steroid was determined at least in duplicate by dextran-charcoal RIA (Hashimoto et a!., 1975). The workable ranges of assay was pg for progesterone and pg for estradiol. Duplicate determinations of FSH and LH in all the plasma samples, minimally at two dilutions, were run simultaneously under the same conditions using NIAMDD-rat-FSH and LH RIA kits; values were expressed in terms of the appropriate NIAMDD-rat- RP-2. The lower limit of assay sensitivity was 15 ng for FSH and 20 ng for LH. The intra- and interassay coefficients of variation for each hormone, calculated from repeated results on pooled samples, varied between 7 and 14%. Statistics Statistical analysis of data was performed by chi-square and Student s t tests. The null hypothesis was rejected when a p value less than 0.05 was obtained. RESULTS Changes in Plasma Progesterone during Metestrous and Diestrous Stages of 4- and 5-Day Estrous Cycles As shown in Figure 1, progesterone in 4-day cyclic rats began to rise at 0900 h and reached its peak level by 2100 h on Day M. By 0100 h on Day D, the steroid returned to a low base line level. Progesterone levels in 5-day cyclic rats were comparable to those in 4-day cyclic animals during Day M. At 0100 and 0500 h on Day D, however, the steroid levels in 5-day cyclic rats remained significantly higher than those in 4-day cyclic ones (p<0.05 and p<0.005, respectively), and thereafter declined to a low value by 1300 h on the same day. Effect of APS Administration on Subsequent Vaginal Cyclicity and Timing of Ovulation Vaginal smear records of individual 5-day cyclic rats given a single injection at 2300 h on Day M of NRS, APS, or APS combined with progesterone are illustrated in Figure 2, where the day of injection is designated as Day 1. Sequential changes in vaginal

3 490 KANEKO ET AL Progesterone - 5-Day cycle - 4-Day cycle a) Cl 0 40 a I I I I IJ I - L/. C,-4 - -,.-4 -I r-.jr- r i #{163}letestrus(M) I I - Lt C. v t- - - Diestrus(D) FIG. 1. Changes in plasma progesterone concentrations during metestrus (M) and diestrus (D) of 4- and 5-day estrous cycles. Time of day is denoted as a 24 h clock. Values are the means and a standard error of 4-7 samples. a: p<0.005; b: p<0.05. smears were not altered in 4 of the 6 NRS-treated rats. In the other 2 rats, however, nucleated smears were observed on Days 3 and 4 respectively, and 1 day of vaginal cornification followed on Day 4 or 5. Thus, 1 of the NRS-treated rats (i6.7%) showed a cornified smear solely on Day 4. On the other hand, 4 of the 7 rats treated with 0.5 unit of APS (57.1%) showed vaginal cornification only on Day 4, and the others had cornified smears on both Days 4 and 5. In 8 of the 10 rats treated with 1.0 unit of APS (80%), 1 day of vaginal cornification followed on Day 3 or 4, although cornified smears were obtained from Day 4 through Day 5 or 6 in the other 2 rats. Four of the 5 rats given progesterone (250 pg) in combination with APS (1.0 unit) showed cornified smears on both Days 4 and 5. Only 1 of the animals (20%) showed vaginal cornification on Day 4 alone. Incidence of 1-day vaginal cornification on Day 3 or 4 (incidence of shortened cycle) was significantly higher in rats treated with 1.0 unit of APS than in rats treated with either NRS or combined APS and progesterone (80% vs. i6.7%, p<0.oo5, X2 8.52, dfi; 80.0% vs. 20%, p<0.05, X2 =5.00, df =1). Another group of 5-day cyclic rats treated with 1.0 unit of APS was autopsied at 1100 h on Day 4 (day of vaginal cornification), and the oviducts were examined for oocytes under a dissecting microscope. The oocytes, ranging from 8 to 12 per rat, were detectable in 3 of the 4 animals. Changes in Plasma Estradiol, FSH, and LH in A PS-Treated Rats As shown in Figure 3, estradiol in control NRStreated 5-day cyclic rats rose gradually from 2300 h on Day M to 1700 h on Day D II. The steroid level at 1700 hon Day DII was significantly higher (p<0.025) than the level at either 2300 h on Day M or 0500 h on Day D. Estradiol levels in the rats treated with 1.0 unit of APS were comparable to those in control animals during Day D. During Day D II, however, levels of the steroid in APS-treated rats remained elevated above the value measured in control animals. Compared to control rats, a significant elevation (p<0.05) was evident at 1100 h in the APS-treated animals. Tonic levels of FSH in control NRS-treated rats showed small but significant rises at 0500 and 2300 h on Day D (0100 h vs h, p<0.oi; 0500 h vs h, p<0.005; iloo h vs h,p<0.05). Antiprogesterone serum administration at 2300 h on Day M resulted in an immediate rise in FSH levels. The hormone levels during Days D and D II, except the

4 DIESTROUS PROGESTERONE IN RAT ESTROUS CYCLES 491 Treatment Vaginal smear record Incidence of shortened cycle(u Day 0 5 NRS 1/6 (l6.7) :I[: Anti-progesterone (APSO.5 unit) :: :: :: : : DpY_0,,,,:,,. V : - : - :.- : -.- I I 4/7 (57.1) Day 0 5 V Anti-progesterone (APS,1.0 unit) II, I - I ll - I - I I I I I I II I : 8/10 (800)ah Anti-progesterone (APS,l.0 unit) & Progesterone(250 g) I I leucocyte Day 0,, 5, - I 1/5 (200)h #{182}9 APS injection (0.5 unit1i.p.) i i nucleated cell comified cell V NRS injection (4.4 ml, i.p.) APS injection (1.0 unit,i.p.) lnection of combined APS 1.0 unit, i.p.) and progesterone (p ; 250 p9, s.c.) FIG. 2. Vaginal smear records in individual rats given a single injection of normal rabbit serum (NRS), antiprogesterone serum (APS), or combined APS and progesterone at 2300 hon metestrus (M). a: p<0.005; b: p<o.os. value at 2300 h on Day D, remained significantly higher (p< ) in APS-treated rats than in control animals. Transient rises in tonic LH levels were also evident at 0500 and 2300 h on Day D in control NRS-treated rats (0100 h vs h,p<0.005;osoohvs. llooh, p<0.005; 1100 h vs h, p<0.05). A marked rise in LH levels was measured within 2 h after APS administration. The hormone levels in APS-treated rats were consistently higher than those in NRS-treated animals, and a significant difference (p<0.005) in LH levels between the two groups of animals was evident at 0100 and 1100 hon Day D. DISCUSSION The 5-day cyclic rats inbred in this laboratory exhibit 2 days of vaginal cornification. Pre-ovulatory excitation of the hypothalamus and ovulation occur around h on the first day of vaginal cornification and between 0200 and 0600 h on the second day of vaginal cornification, respectively (Furudate et al., 1975). Surging releases of gonadotropins are measured in the afternoon of the first day of vaginal estrus (Kaneko, 1980). Thus, the first day of vaginal estrus in the 5-day cycle corresponds to proestrus in the 4-day cycle.

5 492 KANEKO ET AL. pg/mi [St radio! Normal rabbit serum(nrs) Anti -progesterone (APS) NRS or U APS 30 (J ng/mi ng/ml FSH I I I U FIG. 3. Changes in plasma estradiol, FSH, and LH concentrations in 5-day cyclic rats treated with normal rabbit serum (NRS) or antiprogesterone serum (APS). Time of day is denoted as a 24 h clock. Each rat was given a single i.p. injection of either NRS (4.4 ml) or APS (1.0 unit) at 2300 h on metestrus (M). Values are the means and a standard error of 5 samples. a bj: p<0.025; c f n: p<0.05; d k: p<0.01; e g h I 1 m o p: p< The results of the present investigation have replicated the previous finding that progesterone secretion remains significantly higher in the 5-day cyclic rat than in the 4-day cyclic animal during the morning of Day D (Roser and Block, 1969; Furudate et al., 1975; van der Schoot and de Greef, 1976; Nequin et al., 1979). Since suppression of estrus and ovulation by functional corpora lutea and by progesterone administration has been well documented, and since a single dose of the steroid injected during Days M and D in a 4-day cycle delays the subsequent ovulation by 1 or 2 days (Everett, 1948; Brown-Grant, 1969; Weik et al., 1971), it seems plausible that increased progesterone secretion beyond Day M is causally related to recurrence of a 5-day cycle. This view has been substantiated by the following findings in the present investigation. Injection of sufficient antiserum to block the quantities of progesterone present on Day D in a 5-day cycle reduces the duration of ensuing vaginal cornification by 1 day without altering the onset time significantly. The procedure also advances ovulation by 1 day in the majority of animals. Furthermore, progesterone administered with APS eliminates the effect of the antiserum. Within 2 h after APS administration, tonic levels of both FSH and LH increased significantly and remained

6 DIESTROUS PROGESTERONE IN RAT ESTROUS CYCLES 493 consistently higher than the control value. A significant elevation above the control value of plasma estradiol, however, followed as late as 36 h after administration of the antiserum. Therefore, the action of gonadotropins on estrogen secretion is not a process triggered immediately in an all-or-none fashion. Instead, gonadotropins must continue to act for some time for ovarian estrogen secretion to increase. The present findings suggest that the prolongation of luteal progesterone secretion in an estrous cycle suppresses pituitary gonadotropin secretion. Rather than blocking the estrogen triggering of ovulatory LH surge, directly, it delays the estrogen secretion itself, which decreases the threshold of the neural and/or hypophyseal structures for ovulatory LH release. Surge levels of both FSH and LH had been expected to be measured in the afternoon of Day D II in the APS-treated 5-day cyclic rats, since 3 of the 4 animals receiving the same treatment ovulated by 1100 h on the following day. However, none of the rats treated with the antiserum showed the ovulatory increase of these hormones at 1700 h on Day D II. The onset of ovulatory gonadotropin release may have been delayed for several hours in these animals. This delay may be causally related to an insufficient proestrous estradiol level to trigger the properly timed ovulatory release of gonadotropins. Nequin et al. (1979) have observed that estradiol begins to rise at approximately the same time on the day before proestrus in both the 4-day and 5-day cyclic rats, although several reports have suggested that estrogen secretion may begin earlier in the 4-day cyclic rat (Schwartz and Talley, 1965; Shirley et al., 1968; Schwartz and Ely, 1970). The data obtained in this investigation do not rule out the possibility that the threshold to the estradiol trigger is higher in the 5-day cyclic rat. Tonic levels of gonadotropins in the control NRStreated 5-day cyclic rats showed a transient rise at 0500 h on Day D that preceded the lowering of plasma progesterone levels. Injections of sufficient antiovine-lh serum at 1900 h on Day M and 0800 h on Day D in a 5-day cycle resulted in a prolongation of vaginal diestrus (4-6 days), which was accompanied by progesterone secretion elevated above the control value at 1800 h on Day D (Hashimoto and Kawaminami, 1983). In addition, corpora lutea of rats treated with the antiserum can be rescued by exogenously administered prolactin later in their life span (i.e., after ovulation), than can corpora lutea of control animals (Aizawa et al., 1982). Uchida et al. (1969) have suggested that the pituitary is not required for the elevation of progesterone secretion on Day M. These observations may indicate a luteolytic mechanism in corpora lutea of the rat estrous cycle. Tonic levels of LH secreted after ovulation, which stimulates ovarian estrogen secretion, may cause functional luteolysis of a crop of newly formed corpora lutea in the cycle, whereas it has been suggested by Meites et al. (1972) that prolactin induces morphological or structural luteolysis of the earlier crop of corpora lutea during each cycle. Changes in tonic levels of gonadotropins in the 4-day cyclic rats were not determined in the present investigation. Schwartz and Ely (1970), however, have provided evidence suggesting that secretion of LH acting on ovarian estrogen secretion begins earlier in the 4-day cycle than in the 5-day cycle. Therefore, it may be most pertinent to assume that the shorter duration of diestrous 4-day progesterone secretion in the 4-day cycle is due to the earlier imposition of luteolytic action of tonic levels of LH (Hashimoto and Kawaminami, 1983). Several previous investigations have suggested that the onset of ovulatory LH release is more than 1 h earlier in the 5-day cyclic rats than in the 4-day cyclic animal (Hoffman and Schwartz, 1965; Schwartz, 1969; van Rees, 1972). In addition, Smith et al. (1973) have measured higher FSH, LH and prolactin values during proestrus in the 5-day cyclic rat. In this laboratory, however, we measured lower LH and higher prolactin levels during the proestrous afternoon in the 5-day cyclic rat than in the 4-day cyclic animal (Eto et al., 1984). The difference in the ovulatory gonadotropin release in the rats of two cycle lengths could be causally related to a difference in timing of imposition of the luteolytic effect of LH secreted after ovulation and could constitute an important regulatory mechanism by which progestational function of corpora lutea of rat estrous cycles is determined (Hashimoto et al., 1984). ACKNOWLEDGMENTS The authors wish to thank Dr. A. F. Parlow and the Rat Pituitary Hormone Distribution Program, NIAMDD, NIH, USA, for rat-fsh and LH radloimmunoassay kits. Their appreciation is also expressed to Edwin Bruce Jones, M. Ed., for his valuable advice during the preparation of the manuscript, and to Profs. L. L. Anderson, Iowa State University. and K. 1-lomma, University of Tokyo, for critically reviewing the manuscript. REFERENCES Abraham GE, Radioimmunoassay of steroid in biological materials. Acts Endocrinol Suppl 183:7-42 Aizawa K, Seya Y, Hashimoto 1, Effect of anti-ovine-lh serum administration on rescue by prolactin of corpora lutes of rat

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