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Regular Article |
a Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
b School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, United Kingdom
c Air Pollutants Health Effects Research Team, Environmental Risk Assessment Project, National Institute of Environmental Studies, Tsukuba, Ibaraki 305-0053, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ßB subunits) remained very low until PND 15 and then increased by approximately 24-fold. In contrast, plasma levels of inhibin A ( and ßA subunits) were relatively low and steady until PND 20, then increased by approximately 3-fold at PND 25. Changes in ovarian inhibin A and B levels closely resembled those in plasma levels. Plasma FSH levels were low at PND 10 but started to peak from PND 15 and remained high until PND 20, followed by a remarkable reduction at PNDs 25 and 30. This dramatic fall in FSH coincided with the rise of inhibin A. A significant inverse correlation was observed between plasma FSH and plasma inhibin A (r = -0.67, P < 0.0002), ovarian inhibin A (r = -0.48, P < 0.01), plasma inhibin B (r = -0.48, P < 0.05), and ovarian inhibin B (r = -0.54, P < 0.01). Plasma estradiol-17ß levels were elevated from PND 5 through PND 15 , then fell sharply through PND 30. Plasma estradiol-17ß was significantly and positively (r = 0.75, P < 0.0002) correlated with plasma FSH. Plasma LH rose to higher levels at PND 15 and tended to be lower thereafter. The inhibin , ßA, and ßB subunits were localized to primary, secondary, and antral and large antral follicles, but the types of these immunopositive follicles varied with age. It appeared that, at PND 25 and afterward, all three subunits were mainly confined to large antral follicles in the ovary. We conclude that estradiol-17ß likely is the major candidate in stimulation of FSH secretion in the infantile female rat. We also conclude that inhibin regulation of pituitary FSH secretion through its negative feedback in the infantile female rat begins to operate after PND 20. We suggest that this negative feedback is achieved by increases in plasma levels of the two dimeric forms, and that inhibin A appears to be the major physiological regulator of FSH secretion at the initiation of this mechanism. We also conclude that large antral follicles in the ovary are the primary source of these bioactive inhibins that are secreted in large amounts into the circulation after PND 20.
follicle-stimulating hormone, follicular development, granulosa cells, inhibin, mechanisms of hormone action, ovary, pituitary hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inhibins are a group of glycoprotein hormones secreted by the gonads in males and females. The inhibin subunit heterodimerizes with inhibin ß subunits to form inhibin A ( and ßA) and inhibin B ( and ßB), which are the bioactive forms of inhibin in the general circulation. These bioactive forms have been well characterized as negatively regulating pituitary FSH secretion through direct action on the pituitary gonadotrophs in mature female and male rats [9]. In the infantile female rat, plasma immunoreactive inhibin (ir-inhibin) can be detected by RIA as early as PNDs 1–5, and the levels start to rise thereafter [4, 10]. However, the classical negative feedback regulation of FSH by circulating inhibins does not appear to occur during PNDs 10–20, when plasma FSH levels are also elevated [10]. Additionally, injection of anti-inhibin serum, which increases plasma FSH levels in adult females [11], had no effect in the infantile female rat when injected at PND 10 [4]. This led to the hypothesis that the negative feedback regulatory mechanism of circulating inhibins on pituitary FSH secretion begins to work after approximately PND 20–25 in female rats. However, whether the absence of a negative feedback action of inhibins on FSH secretion at an early age in the infantile female rat is due to relatively insensitive pituitary gonadotrophs to circulating inhibins or actually reflects circulating levels of bioactive dimeric forms of inhibin is not yet known. Furthermore, characterization of the ability and the types of follicles in the ovary of the infantile female rat to produce inhibin and two forms of ß subunits may also serve as a good indicator of dimeric inhibins in the circulation. This has been demonstrated previously using an immunohistochemical approach [12, 13], but to our knowledge, a detailed study demonstrating immunolocalization of all three inhibin subunits in the female rat ovary during PNDs 5–30 has not been reported.
Estradiol-17ß plays a key role in the regulation of many pituitary hormones. In the rat, binding of estradiol-17ß to its receptors in the anterior pituitary shows a dramatic increase around PNDs 10–15 [14]. At this time, the expression of estradiol-17ß receptor (ER) ß mRNA is much greater in the pituitary gonadotrophs of prepubertal than in adult female rats [15], and this is probably reflected by higher plasma FSH levels resulting from elevated estradiol-17ß levels in the prepubertal female rat. Additionally, estradiol-17ß directly affects pituitary FSH secretion in 8- to 14-day-old female rats [16]. However, to what degree a casual relationship exists between the two hormones during the prepubertal period from PND 5 to PND 30 is not known in the female rat.
Thus, in the present experiments, we investigated the immunolocalization of all three inhibin subunits in the ovary; ovarian and plasma concentrations of ir-inhibin, inhibin A, and inhibin B; plasma concentrations of estradiol-17ß; and pituitary and plasma concentrations of FSH and LH from PND 5 to PND 30 in female rats. Based on changes in hormonal patterns, relationships between gonadal hormones (i.e., dimeric inhibins and estradiol-17ß) and plasma FSH were also explored.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Wister rats (3–4 mo old) were purchased from Imamichi Institute for Animal Reproduction, Ibaraki, Japan. They were housed in metal cages and maintained in a room with controlled illumination (14L:10D, lights-on at 0500 h) and temperature (22°C), with free access to commercial pellets and tap water ad libitum. They were checked daily for estrous cyclicity by examining vaginal smears, and rats with regular 4-day estrous cycles were selected for mating. The selected female rats were mated with stud male rats that were chosen from our own colony in our animal facility. After mating, females were housed in groups (4–5 rats/cage) until Day 15 of pregnancy, then housed individually until the day of parturition. After birth, only female rat pups were placed with mother rats, with eight to nine pups per mother, and the male pups were killed. Female pups were killed by decapitation at PNDs 5, 10, 15, 20, 25, and 30, and blood was collected. To obtain enough plasma for measurement of hormones, blood from three to five, two to three, and two pups was pooled for each sample at PNDs 5, 10–15, and 20, respectively. A total of 5–11 samples was obtained at each age for subsequent measurement of hormones. The ovaries and pituitaries were removed and weighed. A dissection microscope (K-280; Konan Camera R&I, Hyogo, Japan) was used to remove the ovaries from 5-, 10-, 15-, and 20-day-old pups. All procedures were carried out in accordance with the guidelines established by the Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, for the use of laboratory animals.
Blood and Organ Processing
All blood samples were collected into plastic tubes containing heparin (15 IU/ml blood) as an anticoagulant to prevent clotting. The samples were stored in ice and centrifuged at 1700 x g for 15 min at 4°C immediately after completing the experiment. The resulting plasma was decanted and stored at -20°C until assayed for plasma concentrations of ir-inhibin, inhibin A, inhibin B, estradiol-17ß, testosterone, LH, and FSH.
One ovary from each rat was collected for measurement of ovarian inhibins. Each sample was obtained by homogenizing 10 (at PND 5) or 4 (at PNDs 10 and 15) ovaries per tube in 0.5 ml or 2 ovaries (at PNDs 20, 25, and 30) per tube in 1 ml of 0.85% (w/v) NaCl. The pituitary glands (one per tube) were homogenized in 1 ml of 0.85% (w/v) NaCl. The homogenized samples (Ultrasonic Disruptor, Model UR-200P; Tomy Seiko Co., Ltd., Tokyo, Japan) were centrifuged at 15 000 x g for 30 min at 4°C. The supernatant was decanted and stored at -20°C until assayed for ovarian ir-inhibin, inhibin A, and inhibin B and pituitary LH and FSH concentrations.
The other ovary was fixed in 4% (w/v) paraformaldehyde for 24 h and then dehydrated using a series of graded alcohol and embedded in paraffin. Tissue sections (thickness, 4 µm) were cut using a microtome and mounted onto poly-L-lysine-coated (0.01%, w/v) slide glasses, dried overnight at 32°C, and prepared for histological and immunohistochemical examinations.
Radioimmunoassay
Plasma and ovarian concentrations of ir-inhibin were measured by a double-antibody RIA as described previously [17]. Purified bovine 32-kDa inhibin was used as standard. The assay system does not distinguish dimeric inhibin from the subunit monomer. Intra- and interassay coefficients of variation were less than 11%.
Plasma concentrations of estradiol-17ß and testosterone were determined by double-antibody RIAs using 125I-labeled radioligands as described previously [18]. Antibodies against estradiol-17ß (GDN 244) and testosterone (GDN 250) were kindly supplied by Dr. G.D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO). Intra- and interassay coefficients of variation were less than 20%.
Plasma and pituitary concentrations of LH and FSH were measured using National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; Baltimore, MD) RIA kits for rat LH and FSH. The antiserum used was anti-rat LH (S-10) and anti-rat FSH (S-11). Intra- and interassay coefficients of variation for LH and FSH were less than 12%.
ELISA of Inhibin A and Inhibin B
Plasma and ovarian concentrations of inhibin A and inhibin B were determined using a two-site ELISA specific for each peptide as previously described [19]. Intra- and interassay coefficients of variation for inhibin A and inhibin B were less than 6%.
Histology and Immunohistochemistry
The ovarian tissue sections from rats of all ages were stained with hematoxylin-and-eosin for histological examination of growing follicles. The tissue sections were deparaffinized with xylene, passed through a graded series of alcohol, and prepared for immunohistochemical staining. The antibody against each inhibin subunit was anti-[Tyr30]inhibin--chain (1–30)-NH2 conjugated to rabbit serum albumin, anticyclic inhibin ßA (81–113)-NH2, and anticyclic inhibin ßB (80–112)-NH2. The inhibin subunit peptide was kindly provided by Dr. N. Ling (Neurocrine Biosciences, Inc., San Diego, CA) and the anticyclic inhibin ßA (no. 305-24-D) and anticyclic inhibin ßB (no. 305-25-D) by Dr. W. Vale (Salk Institute for Biological Studies, La Jolla, CA). The use of these antibodies against inhibin subunits has been previously described [20]. Following the completion of various steps as described previously [21], the sections were incubated overnight at 37°C with the respective polyclonal antibody at a dilution of 1:20 000 (for inhibin and ßB) or 1:80 000 (for inhibin ßA) in 0.5% (w/v) casein-Tris saline (0.05 M Tris-HCl with 0.15 M NaCl, pH 7.6). The second antibody used was biotinylated goat anti-rabbit antibody (ABC Kit Elite; Vector Labs, Burlingame, CA) at 0.25% (v/v), followed by the addition of avidin-biotin at 2% (v/v). The primary antibody bound to the sections was visualized by treatment with 0.025% (w/v) 3,3'-diaminobenzidine tetrachloride (Sigma Chemical Co., St. Louis, MO) in 10 mM Tris-buffered saline containing 0.01% (v/v) H2O2 for 1–2 min. Specificity of the antibodies was examined using normal rabbit serum instead of primary antibody using a 1:20 000 dilution. All tissue sections were counterstained with hematoxylin for 15 sec.
Statistical Analyses
Means between different age levels of individual hormone were compared using ANOVA and least-squares means generated using the general linear model procedure of the Statistical Analysis System (SAS) [22]. Canonical correlation analysis was performed to measure correlation between the individual variables, and statistical significance was obtained using the Wilks 
test. Data are presented as the least-square mean ± SEM. A probability value (P) of less than 0.05 was considered to be significant. All statistical analyses were carried out using the SAS computer package.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma ir-inhibin was detectable as early as PND 5, and the concentrations were relatively low until PND 15, then progressively increased through PND 30. Thus, plasma ir-inhibin concentrations found at PND 25 (P < 0.01) and PND 30 (P < 0.001) were significantly higher compared with those at PND 15 (Fig. 1A). The pattern of changes in ovarian concentrations of the hormone was similar to that in plasma concentrations, except that ovarian ir-inhibin was elevated to much higher levels at PND 10 (Fig. 1B). Plasma inhibin A concentrations changed from PND 5 to PND 30 in a fashion similar to that of ir-inhibin. The most striking increase of plasma inhibin A levels occurred from PND 20 to PND 25 and represented a threefold increase (P < 0.0002) compared with the value at PND 20 (Fig. 1C). The levels continued to increase (P < 0.05) through PND 30. Changes in ovarian inhibin A concentrations were somewhat similar to those in plasma levels but continued in a fashion identical to that of ovarian ir-inhibin concentrations, with a similar marked increase at PND 10 (Fig. 1D). Plasma inhibin B was undetectable at PND 5; thereafter, inhibin B was detectable in plasma, but at very low levels until PND 15 (Fig. 1E). This was followed by a dramatic rise in concentration, leading to a 24-fold increase (P < 0.0002) by PND 20 compared with the value at PND 15. The concentrations did not significantly increase thereafter. Ovarian inhibin B concentrations followed a somewhat similar pattern to those of plasma inhibin B but showed a significant (P < 0.01) increase at PND 10, as seen with ovarian inhibin A and ir-inhibin concentrations. The concentrations found at PNDs 20 and 25 (P < 0.05) and at PND 30 (P < 0.0002) were significantly higher compared with the value at PND 15 (Fig. 1F). Interestingly, a significant reduction in the ovarian levels of all three forms of inhibins, such as ir-inhibin (P < 0.001), inhibin A (P < 0.001), and inhibin B (P < 0.05), was observed from PND 10 to PND 15. However, the changes in ovarian levels of the hormones were reflected in the plasma levels of inhibin A only (P = 0.0627) (Fig. 1C). The ovarian levels of inhibin A and inhibin B, but not of ir-inhibin, at PND 15 were not significantly different from their respective levels at PND 5.
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Plasma estradiol-17ß concentrations were low at PND 5 but increased significantly (P < 0.0002) through PND 10 and 15. This was followed by a dramatic reduction in concentrations through PND 20 (P < 0.0002), and the levels continued to fall until PND 30 (Fig. 2A). Plasma testosterone was lowest at PND 5. Thereafter, the concentrations increased significantly (P < 0.0002) through PND 15 and PND 25, followed by a dramatic fall (P < 0.0002) through PND 30 (Fig. 2B).
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Pituitary Hormones During the Neonatal Period
Plasma FSH concentrations were relatively low at PND 5 and did not change significantly through PND 10. Thereafter, a more-than-twofold increase was found at PND 15, and this level was maintained until PND 20 (Fig. 3A). A striking reduction (P < 0.0002) in the concentrations was found at PND 25, which represented a more-than-ninefold decrease from those found at PND 20. The concentrations at PND 25 and afterward were the lowest observed compared with levels at PNDs 5 and 10 (P < 0.0002). Pituitary concentrations of FSH changed in a very similar fashion, in which a dramatic increase was found from PND 15 to PND 20, followed by a significant (P < 0.0002) reduction through PNDs 25 and 30 (Fig. 3B). Plasma LH concentrations were low at PND 5 and then increased significantly (P < 0.01) through PND 15 (Fig. 3C). Thereafter, the concentrations did not vary significantly through PND 30. Similarly, pituitary LH concentrations were significantly (P < 0.0002) increased through PND 20 and remained elevated thereafter (Fig. 3D).
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Correlations Between Ovarian and Pituitary Hormones
Plasma inhibin A levels were significantly and negatively correlated (r = -0.67, P < 0.0002) with plasma levels of FSH during the period from PND 5 to PND 30 (Fig. 4A). A significant negative correlation (r = -0.48, P < 0.01) was also found between ovarian inhibin A and plasma FSH concentrations (Fig. 4B). Similarly, plasma inhibin B levels were significantly and negatively correlated (r = -0.48, P < 0.05) with plasma levels of FSH during the period from PND 5 to PND 30 (Fig. 4C). Furthermore, the ovarian concentrations of inhibin B were also significantly and negatively correlated (r = -0.54, P < 0.01) with plasma FSH concentrations (Fig. 4D). Additionally, a highly significant positive correlation (r = 0.75, P < 0.0002) was observed between plasma levels of FSH and estradiol-17ß (Fig. 4E). A significant positive correlation (r = 0.34, P < 0.05) between plasma levels of LH and testosterone during the period from PND 5 to PND 30 was observed as well (Fig. 4F).
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Histology and Immunohistochemistry
At PND 5, many of the small primordial/primary follicles in the ovary stained strongly positive for the inhibin subunit (Fig. 5A). Many of these follicles also stained positive for the inhibin ßA (Fig. 5B) and ßB (Fig. 5C) subunits, but the staining specificity was less prominent compared with that of the inhibin subunit.
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At PND 10, granulosa cells surrounding small primary and secondary follicles stained strongly positive for all three inhibin subunits (Fig. 5, E–G).
At PND 15, many follicles, including primary, secondary, and those with developing antrum, stained positive for the inhibin subunit (Fig. 5I). Immunopositive staining for both inhibin ßA (Fig. 5J) and ßB (Fig. 5K) subunits was observed, but apparently, fewer follicles stained positive compared with what was observed for the inhibin subunit. However, it appeared that the intensity of the immunostaining reaction of the inhibin ßB subunit was somewhat higher than that of the inhibin ßA subunit, particularly in small antral follicles.
At PND 20, many follicles of all types, including antral follicles, stained strongly positive for the inhibin subunit (Fig. 5M). In contrast, the immunostaining reaction for the inhibin ßA (Fig. 5N) and ßB (Fig. 5O) subunits appeared to be localized primarily to granulosa cells of antral follicles and to some secondary follicles. The immunopositive reaction did not appear to be localized to primary follicles. It also appeared that the immunostaining reaction for the inhibin ßA and ßB subunits was somewhat more intense in antral than in secondary follicles.
At PND 25, large antral follicles were first seen. These large antral follicles were the main types of follicles that stained strongly positive for the inhibin subunit (Fig. 5Q). However, specific staining was also observed in other types of follicles, with primary follicles being the least stained. In comparison, large antral follicles were the major type of follicles that showed a strongly positive immunostaining reaction for both inhibin ßA (Fig. 5R) and ßB (Fig. 5S) subunits. Unlike the case with inhibin subunit staining, inhibin ßA and ßB subunits did not appear to stain positive in primary and secondary follicles but did in small antral follicles.
At PND 30, the pattern of immunostaining reactions for all three inhibin subunits was very much similar to that observed at PND 25. However, the number of large antral follicles that stained positive for the inhibin subunit (Fig. 5U) was apparently not affected at PND 30, yet the number of those that stained strongly positive for both inhibin ßA (Fig. 5V) and ßB (Fig. 5W) subunits appeared to have increased by PND 30.
For comparison, ovarian sections incubated with normal rabbit serum instead of primary antibody to examine the antibody specificity at PND 5 (Fig. 5D), 10 (Fig. 5H), 15 (Fig. 5L), 20 (Fig. 5P), 25 (Fig. 5T), and 30 (Fig. 5X) are also shown.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dimeric inhibins are the bioactive forms of inhibin that, through negative feedback action, regulate pituitary FSH secretion in the adult female rat [23]. Although previous studies have elaborated the importance of inhibins in the regulation of FSH secretion, particularly during the estrous cycle of female rats [23, 24], no report to date has demonstrated how dimeric inhibins are involved in the regulation of FSH secretion during early postnatal life in the female rat. A significant rise in FSH levels occurs around PND 10, followed by a rapid decline by approximately PNDs 20–25 [1–5]. The number of follicles that start to grow per day in 16-day-old rats is reportedly higher than that in older rats [25]. Thus, previous reports indicate that the elevation of FSH during this time appears to be essential for normal follicular development in the neonatal rat and mouse [6–8]. The present data are in agreement with those of previous studies that reported elevated plasma FSH levels during the second to third week of postnatal life in the female rat.
Previous reports indicated that inhibin does not appear to regulate plasma FSH until around PND 20 in the female rat [2–4, 10]. Therefore, it has been suggested that inhibin regulation of FSH is not operational due to a lack of sensitivity by the pituitary gonadotrophs to gonadal feedback during early postnatal period. These conclusions were based on the observations that ovariectomy or injection of inhibin antiserum were not effective in causing changes to the plasma FSH level in infantile rats aged less than 15 days [3, 4]. Those investigators measured immunoreactive inhibins, but not the bioactive forms, by RIA systems that failed to distinguish dimeric inhibin from the subunit monomer. This is presumably because a specific, sensitive assay to measure dimeric forms was not available at that time.
We found, however, that levels of the dimeric forms of inhibin were relatively low from PND 5 until PND 20 and then begin to increase through PND 30. It is important to note that a significant reduction in the ovarian levels of all three inhibin forms at PND 15 might have occurred as a result of elevated estradiol-17ß levels. A recent study demonstrated that granulosa cell cultures prepared from the ovaries of 25-day-old rats pretreated with diethylstilbestrol for 4 days produced significantly reduced levels of both inhibin ßA and ßB subunit mRNAs compared with those produced by granulosa cell cultures from untreated rats [26]. In line with this, we found fewer follicles staining positive, together with a somewhat reduced immunostaining intensity, for both inhibin ßA and ßB subunits in the ovaries of 15-day-old rats. On the other hand, ovarian ir-inhibin levels also fell significantly at PND 15, which is in contrast to the findings of the above study [26] that showed no effect on inhibin subunit mRNA expression from diethylstilbestrol treatment. Only inhibin A and inhibin B levels were maximally affected, however, because the levels of the two hormones at PND 15 were not significantly different from those at PND 5. Furthermore, the present results appear to agree with those of Drummond et al. [26], who showed that the expression of all three inhibin subunit mRNAs in the ovaries increased from 4 to 8 days of age, followed by a significant reduction in the ovaries at 12 days of age. In contrast, we found that changes in ovarian levels of the hormones did not affect plasma levels, except for inhibin A, which was marginally affected.
Inhibin B did not suppress plasma FSH levels despite its 24-fold increase at PND 20. Nevertheless, the dramatic reduction in plasma FSH that occurred at PND 25 was associated with a threefold increase in plasma inhibin A levels, which continued to rise thereafter. Furthermore, plasma inhibin B levels at this time were not significantly different from the preceding values at PND 20. It was also evident that, at PNDs 20 and 25, plasma inhibin B levels were quantitatively well below those observed for plasma inhibin A. However, such a comparison may not be possible, because a precise comparison, both qualitatively and quantitatively, of inhibin A and inhibin B bioactivities using recombinant material in rat cultured pituitary cells has not yet been reported. Both forms of dimeric inhibin extracted from human follicular fluid and serum appeared to be equally potent in attenuating FSH secretion in the rat pituitary cells using in vitro bioassay systems [27]. This is probably reflected by the findings of the present study that ovarian and plasma concentrations of both forms of dimeric inhibin were negatively correlated with plasma FSH concentrations. Previous reports have suggested, however, that inhibin regulation of FSH also depends on the actual levels of inhibin in the circulation [2]. Thus, the remarkable fall in plasma FSH levels that started to occur after PND 20 and the concomitant rise of plasma inhibin A suggest that inhibin A rather than inhibin B likely is the major candidate for the physiologically important form of dimeric inhibin at the initiation of the FSH-suppression mechanism in infantile female rats. In line with this, we found that the significance of the correlation between plasma inhibin A and FSH was very prominent compared with that observed for plasma inhibin B versus FSH. Furthermore, the findings that injection of steroid-free bovine follicular fluid (bFF) decreased plasma FSH levels 8 h later in intact, 15-day-old female rats, and that bFF injection prevented the rise in FSH in rats of the same age that were ovariectomized 2 days before [2], suggest that the pituitary gonadotrophs are still sensitive to a direct action of bioactive inhibins during early infantile life. Thus, our data clearly suggest that inhibin regulation of FSH does not occur before PND 20, because the circulating concentrations of bioactive dimeric forms of inhibin are low during this period in the infantile female rat. Additionally, the difference in concentrations between ir-inhibin and inhibin A and/or inhibin B could be due to a relatively higher level of circulating free subunit [26].
In the present study, the rise in plasma FSH levels and the rapid decline a few days later closely resembled those of the pituitary FSH levels. The changing patterns of plasma FSH and of estradiol-17ß in the present study agree with the findings of earlier studies [1], including that the highest estradiol-17ß levels coincided with the maximal FSH levels, which occurred at PND 15. Estradiol-17ß plays a key role in the regulation of many pituitary hormones. In the present study, plasma estradiol-17ß likely is the major endocrine hormone involved with stimulation of pituitary FSH secretion in the infantile female rat. Wilson et al. [15] reported that the expression of ER ß mRNA was much greater in the pituitaries of prepubertal than in those of adult female rats, and that the expression of ER ß was doubled in the pituitaries of 15-day-old female rats. Additionally, approximately 84% of pituitary FSH-immunopositive cells expressed ER ß. Administration of the estradiol-17ß-antagonist tamoxifen into infantile female rats during a 3-day period, either at PNDs 8–11 or 11–14, resulted in significantly suppressed FSH secretion [16]. Wilson and Handa [16] also characterized the stimulatory effects of estradiol-17ß on FSH secretion in vitro. Thus, the highly significant positive correlation observed between plasma estradiol-17ß and FSH levels in the present study indicates that elevated levels of estradiol-17ß might be responsible for the rise in FSH secretion seen in these infantile rats, perhaps through stimulation of activins produced locally in the pituitary gonadotrophs [28]. A greater reduction in plasma estradiol-17ß levels after PND 15 was accompanied by a marked decrease in plasma FSH levels after PND 20. This reduction of plasma estradiol-17ß probably reflects cessation of the adrenal gland contribution of estradiol-17ß, because this organ is known to contribute a significant proportion of plasma estradiol-17ß until approximately PNDs 10–15 in the female rat [29].
Although the granulosa cells of developing follicles may be expected to continually produce estradiol-17ß, plasma levels were decreased instead. Activin A stimulates progesterone production in a dose-dependent manner in ovarian cell cultures from 4-, 8-, and 12-day-old rats in the presence of FSH [12]. Doses of FSH used in that study as well as in another [30] using 4-day-old rat ovary did not enhance progesterone production when given alone, and varying doses of activin A also had no effect on progesterone production when given alone [12]. These data suggest that activin A stimulates progesterone synthesis and, perhaps, production of other steroids, but this appears to occur only in the presence of FSH. In line with this, activin A also stimulates production of mRNAs for cytochrome P450 cholesterol side-chain cleavage enzyme in FSH-stimulated granulosa cells from immature rats [31] and enhances FSH-induced activity of aromatase and production of progesterone in nondifferentiated granulosa cells in vitro [32]. Thus, the observed reduction in plasma estradiol-17ß levels in rats during the present study may be due to a markedly reduced FSH secretion that, in turn, results from a negative feedback of elevated dimeric inhibin levels.
Our immunohistochemical data suggest that, from as early as Day 5 after birth, ovarian/granulosa cells have the ability to synthesize all three forms of inhibin subunit proteins, and our data agree with the findings of other investigators [12] who showed that inhibin , ßA, and ßB subunit proteins and mRNA for and ßA subunits are localized to the follicular tissues in the ovary of 4-, 8-, and 12-day-old infantile female rats. In the present study, relatively higher levels of plasma inhibin A, but not of inhibin B, were detected in 10- to 20-day-old infantile rats at a time when FSH levels were also elevated. Plasma FSH has been shown to be a major stimulus for inhibin production by ovarian granulosa cells in vitro [12, 33, 34]. Dispersed ovarian cells from 8- and 12-day-old rats produced inhibin in a dose-dependent manner in response to FSH [12]. The dimerization process of the subunit with either of the two ß subunits in the infantile female rat appears to be regulated differentially, because inhibin B levels are reportedly higher than inhibin A levels in the plasma of adult female rats [23, 35]. Welt and Schneyer [34] reported that human preantral follicle cultures produced detectable levels of inhibin B, but not of inhibin A, in response to FSH treatment. On the other hand, human antral follicle cultures produced detectable amounts of both inhibin A and inhibin B. In the present study, antral or antral cavity-forming follicles were first seen in the ovaries of 15-day-old or older rats, as has also been observed by others [36], yet inhibin A levels were already high in both the plasma and ovary of 10-day-old rats. This suggests that, in the infantile female rat, the regulatory mechanisms involved in the synthesis and secretion of dimeric inhibins into the circulation differ from those in the adult female. In addition, inhibin B concentrations were relatively high and measurable in the ovary of 5- to 15-day-old rats, but plasma levels were less than the detection limit at PND 5 and very low at PNDs 10 and 15, suggesting a differential regulation of the secretion of each dimer. Furthermore, our data agree with the findings in recent studies that cultures of granulosa cells obtained from 24- to 25-day-old female rats [33] or ovarian cell cultures of 4-, 8-, and 12-day-old rats [26] consistently produced more inhibin A than inhibin B in response to FSH stimulation. In the former study [33], addition of activin A, the homodimer of the ßA subunit, differentially stimulated the production of inhibin B more than the production of inhibin A, and the stimulatory effect on inhibin B was more pronounced in the presence of exogenous FSH. Our immunohistochemical data indicate the possibility of the formation of activin dimers, although we did not measure plasma levels of activins in the present study. We found that plasma levels of inhibin B, but not of inhibin A, increased by approximately 24-fold from PND 15 to PND 20 at a time when plasma FSH had reached its highest level. This raises the possibility that activin A might be the factor that mediated this sharp increase of inhibin B observed at PND 20.
The pattern of changes in plasma LH levels was similar to that reported previously [1]. A considerable variation in LH levels observed among the neonates is probably due to a diurnal fluctuation of its release, as suggested by Dohler and Wuttke [1]. Plasma LH reached its maximum levels at PND 15, followed by a tendency to decrease through PND 30. The first peak of plasma testosterone coincided with the peak of plasma LH at PND 15. Indeed, hCG stimulated testosterone production by 10-day-old rat ovaries [37], whereas LH stimulated androstenedione production by cultured ovarian follicles from immature rabbits as well as from mature rats [38, 39]. It has also been reported that LH can augment progesterone production by cultured ovaries from 4-day-old rats after incubation for more than 4 days [30], that LH can increase cAMP production in juvenile ovaries incubated in vitro [40], and that exogenous dibutyryl cAMP not only can increase progesterone production but also the activity of another steroidogenic enzyme, aromatase, which is the key enzyme involved in estrogen biosynthesis [30]. Thus, in the present study, LH might be the major stimulus for testosterone production, and this may also be reflected in the positive correlation observed between the two hormones.
In summary, we conclude that estradiol-17ß likely is the major candidate in the stimulation of FSH secretion in the infantile female rat. We also conclude that inhibin regulation of pituitary FSH secretion in the infantile female rat begins to operate after PND 20. This negative feedback is achieved by increases in the plasma levels of dimeric inhibins, but inhibin A likely is the major candidate at the initiation of this mechanism. We also suggest that large antral follicles are mainly responsible for the synthesis and secretion of bioactive inhibins in large amounts into the circulation.
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ACKNOWLEDGMENTS |
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(1–30) peptide, Dr. G.D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO) for the anti-testosterone (GDN 250) and anti-estradiol (GDN 244), and Dr. W. Vale (Salk Institute for Biological Studies, La Jolla, CA) for the anticyclic inhibin ßA (81–113; no. 305-24-D) and anticyclic inhibin ßB (80–112; no. 305-25-D). We also thank Mr. K. Ohshima from our laboratory for his excellent technical assistance with immunohistochemistry and ELISA. |
FOOTNOTES |
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1 Supported by a grant-in-aid for Scientific Research from the Ministry of Education of Japan. 
2 Correspondence: Kazuyoshi Taya, Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. FAX: 81 42 367 5767;taya{at}cc.tuat.ac.jp
Accepted: July 5, 2001.
Received: April 26, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-, ßA-, and ßB-subunits in ovarian cell types during the rat estrous cycle. Mol Endocrinol 1998; 2:1352-1363[Abstract]
5/4-isomerase and cholesterol side-chain cleavage cytochrome P450 by activin in rat granulosa cells. Endocrinology 1995; 136:3247-3252[Abstract]This article has been cited by other articles:
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S. Mazaud, C. J. Guigon, A. Lozach, N. Coudouel, M. G. Forest, H. Coffigny, and S. Magre Establishment of the Reproductive Function and Transient Fertility of Female Rats Lacking Primordial Follicle Stock after Fetal {gamma}-Irradiation Endocrinology, December 1, 2002; 143(12): 4775 - 4787. [Abstract] [Full Text] [PDF]
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