Functional Maturation of the Pituitary-Gonadal Axis in the ... - CiteSeerX

BIOLOGY OF REPRODUCTION 52, 1404-1409 (1995)

Functional Maturation of the Pituitary-Gonadal Axis in the Neonatal Female Rat' TARJA A- SOKKA and ILPO T. HUHTANIEMI 2 Departmentof Physiology, University of Turku, 20520 Turku, Finland ABSTRACT The developmental onset of the pituitary feedback response to LH/hCG-stimulated ovarian activity was studied in the neonatal rat. A single injection of hCG (600 IU/kg BW) was administered to groups of rats between I and 10 days of age, and the responses, i.e., ovarian estradiol (E,), progesterone (P), and testosterone (T) production as well as serum LH were monitored 3 days later. The first significant increase in ovarian T and E2 contents occurred in rats treated on Day 8 of life, and the first significant increase in P content occurred when hCG was administered at the age of 9 days. A significant decrease in serum LH, 3 days after hCG injection, was observed for the first time in animals treated on Day 7, but not in those aged 1-6 days. To study whether the appearance of the ovarian response to LH is dependent on FSH, rats received combined treatment with recombinant human (rec)FSH on Days 3-8 (0.3 IU s.c. twice daily) and a single injection of hCG (600 IU/kg BW s.c.) on Day 6. The elevated FSH levels from Day 3 onward advanced the suppression of the serum LH level after hCG injection, suggesting that enhanced action of FSH promotes the appearance of functional LH receptors neonatally. It was concluded that 1) the negative feedback of ovarian activity on pituitary LH secretion is functional in the neonatal rat from Day 10 of life; 2) increased intraovarian T levels reflect the androgenic dominance of this early feedback action; and 3) elevated postnatal levels of FSH may advance the onset of the ovarian response to LH/hCG stimulation.

INTRODUCTION The developmental timing of the onset of pituitary-gonadal feedback regulation in the female rat is still poorly characterized. The ovarian responsiveness to gonadotropins starts at the end of Week 1 of life [1, 2], which agrees well with the onset of gonadotropin binding, that of FSH on Days 4-7 [3,4], and that of LH on Days 5-9 [4-8]. The expression of full-length mRNA for the FSH receptor on Day 1 [9] and for the LH receptor on Day 7 postpartum [10] confirms these findings. Circulating levels of gonadotropins are elevated in the neonatal female rat during the first 2 wk of life [11, 12]; this has been explained by the missing gonadal negative feedback link. In fact, existing data on the onset of estradiol (E 2)-mediated negative feedback in the female are controversial. Ovariectomy during the first postnatal week did not increase serum gonadotropin levels [13]; but when ovariectomy was performed at the age of 10 days, an increase was noticed 2 days later in FSH and LH levels [14, 15]. There are also data showing that the pituitary-gonadal feedback regulation is not fully active before the age of 20 days [16]. ao-Fetoprotein is a confounding factor in the neonatal rat up to about 25 days of age; because it binds to E2 [14,17], it is difficult to assess the extent to which the estrogen produced by neonatal ovaries is biologically active. Moreover, gonadotropin secretion is assumed to be under preferential androgen control during the neonatal (1-7 days) period and partially during the infantile (8-21 days) period Accepted February 1, 1995. Received November 28, 1994. 'This work was supported by a research contract from the Academy of Finland and by a grant from the Sigrid Juselius Foundation. 2Correspondence. FAX: +358-21-2502610.

[15], and the response to the inhibitory signal of E2 matures gradually during the infantile period. The appearance of ovarian LH receptors during adult follicular development is an FSH-dependent phenomenon [18,19], and the role of FSH in the induction of LH receptors has been postulated. Since the onset of FSH action in the neonatal ovary precedes that of LH [4], it is also possible that FSH has a role in the postnatal induction of LH receptors. The first aim of the present study was to detect the time of onset of the suppressive effect of hCG-stimulated ovarian activity on pituitary LH secretion. This study was made possible by a new immunofluorometric assay for rat LH that does not cross-react with hCG and has over 50-fold increased sensitivity in comparison to conventional RIA of this hormone [20]. The second aim was to study whether postnatal elevation of the physiological FSH levels would advance the appearance of LH action in the ovary. MATERIALS AND METHODS Animals and Experimental Design Rats of the Sprague-Dawley strain were used. Newborn rats were combined into litters of 10-12 female pups per lactating mother, and weaning took place at the age of 21 days. The day of birth was designated postnatal Day 1. Human CG Treatment. A single dose of hCG (3000 IU/mg, Pregnyl; Organon International BV, Oss, The Netherlands), 600 IU/kg BW, was injected s.c. (50-200 pl/dose) into neonatal (1-7 day old), infantile (8-10 day old), juvenile (23 day old), and adult (120-180 day old) female rats. The dosage selected was based on earlier dose response studies of the effects of hCG on neonatal rat gonads [21]. Controls received identical injections of saline. The rats 1404

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MATURATION OF THE PITUITARY-GONADAL AXIS

were killed 3 days after hCG treatment by heart puncture under general anesthesia with avertin [22]. The blood collected by heart puncture was centrifuged, and serum was separated and stored at -20°C. The ovaries were removed and immediately snap-frozen in liquid nitrogen and stored at -70 0C until processed for hormone measurements. For the time course study, a single injection of hCG (600 IU/ kg BW) s.c. was administered to 10-day-old rats, which were killed at the age of 11, 12, or 13 days, and to 7-day-old rats, which were killed on the following day. The serum and ovaries were collected and stored as above. CombinedFSH and hCG treatment. Three-day-old rats received twice-daily injections of 0.3 IU (50 l/dose) of recombinant human FSH (recFSH; Org 32489, approximately 10 000 IU/mg; Organon) s.c. until Day 8 of life. The dosage given was selected on the basis of earlier studies using recFSH in rats [23, 24]. The animals also received a single injection of hCG, 600 IU/kg BW s.c., on Day 6 or an identical injection of saline. Controls received identical injections of saline on Days 3-8, and on Day 6 they also received either a second injection of saline or a single injection of hCG (600 IU/kg BW) s.c. The rats were killed 3 days after hCG treatment, and serum and ovaries were collected and stored as above. Hormone Measurements Serum LH was measured through use of an in-house immunofluorometric assay based on the Delfia principle (Wallac Oy, Turku, Finland) [20]. The sample volume used was 25 pl. The sensitivity of the assay was 0.75 pg/tube; the intraassay coefficient of variation (CV) was 8%; and the interassay CV was 12.5%. The cross-reactivity of the assay with hCG is below 0.01%. The results are expressed in terms of the NIDDK (Baltimore, MD) RP-2 standard. For E2, progesterone (P), and testosterone (T) measurements, the ovaries were homogenized in 200 lI of Dulbecco's PBS, pH 7.4. The E2 content was measured by RIA, using an antiserum (code 0341) purchased from Orion-Farmos Diagnostica (Oulunsalo, Finland), after extraction with diethyl ether. P and T were measured by RIAs as described previously [25, 26]. The E2 assay had a sensitivity of 2 fmol/ tube, and the intra- and interassay CVs were less than 15%. The sensitivity of the P assay was 4 fmol/tube; the intraassay CV was 10%, and the interassay CV was 15%. The T assay had a sensitivity of 1 fmol/tube; the intraassay CV was less than 6%, and the interassay CV less than 12%. Ten microliters of the ovarian homogenate was used for protein measurement according to the method of Bradford [27]. StatisticalAnalysis The data were examined by one-way analysis of variance and Duncan's New Multiple Range test. Statistical analyses of hormonal data were performed after logarithmic trans-

formation, and a nonparametric statistical test (Mann-Whitney U-test) was used to analyze the serum LH data because of their skewed distribution. The limit of significance selected was p < 0.05. RESULTS Human CG Treatment The time course of changes in serum LH levels and in steroidogenic responses to hCG stimulation in vivo was monitored in rats injected with hCG (600 IU/kg BW) at the age of 10 days and killed at the age of 11, 12, or 13 days. The decrease in serum LH was not observed before 3 days after the hCG injection, on Day 13 (p < 0.01, Fig. la), despite the clear changes observed earlier in ovarian steroidogenesis. The highest E2 responses to hCG (22-fold) were seen on Day 12 (Fig. lb). The hCG treatment increased ovarian P contents about 10-fold on Day 13 (Fig. Ic). The highest increase occurred in the ovarian T content with a maximum, on Day 12, of about 60-fold (Fig. ld). On the basis of these data, the decision was made to monitor the age-dependent effects of the hCG injection on Day 3 of treatment in subsequent experiments. The effect of hCG on steroidogenesis was also tested in rats treated on Day 7 and killed on the following day. The ovarian E2 and T contents increased 3.5-fold and 10-fold, respectively (Table 1). However, when these steroids were measured on Day 10, the differences were no longer significant (Fig. 2, b and d). Serum LH levels did not differ between control rats and those injected with hCG (600 IU/kg BW, single injection) when the hormone treatment was given at ages 1-6 days and LH was measured 3 days later (Fig. 2a). A statistically significant decrease (p at least < 0.05) in serum LH concentrations occurred for the first time in animals treated at the age of 7 days (i.e., LH measurement on Day 10) and in all older age groups. The decrease in the 7-10-day group was about 50%, from 1.16 + 0.42 to 0.59 + 0.33 RLg/L (p < 0.001), and the suppression was greater thereafter. The ovarian steroidogenic response to hCG in these animals was monitored through measurement of the contents of E2 , P, and T. Human CG increased ovarian E2 contents significantly after Day 11, at which age the stimulation was 6.2-fold (p < 0.001, Fig. 2b). The basal E2 levels remained

TABLE 1. Serum concentrations of LH and intraovarian contents of E2, P, and T in female rats treated with a single injection of hCG (600 IU/kg B.W. s.c.) on Day 7 and killed on Day 8'. Serum LH (hlg/L)

E2 (fmol/ovary)

P (fmol/ovary)

T (fmol/ovary)

Controls 0.35 + 0.12 16.60 + 2.8 26.30 + 2.20 6.38 + 4.01 hCG treated 2 0.24 + 0.03 56.58 11.9* 22.36 2.22 68.12 + 14.00* 'Control and hCG treatment group consisted of 3-5 animals (mean SEM). 2.p < 0.05, **p < 0.01 vs. controls.

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FIG. 1. Time course of response of serum LH (a) and ovarian contents of E2 (b), P (c), and T (d) to treatment of 10-day-old female rats with hCG (600 IU/kg BW, single injection). Controls were treated with vehicle only. The animals were killed after 1, 2, and 3 days, at ages indicated on the x-axis. Values are mean - SEM of measurements from 7-9 animals. **p < 0.01, ***p < 0.001 vs. controls (Mann-Whitney U-test for serum LH, one-way ANOVA after logarithmic transformation for ovarian steroids).

low in the group aged 8-13 days (< 70 fmol/ovary), increasing to 147 ± 21.0 fmol/ovary at the age of 26 days. In randomly cycling adult ovaries, the hCG-induced increase in E2 was about 5-fold (p < 0.001). Ovarian P contents increased in hCG-treated rats, compared to controls, after Day 12 (Fig. 2c). At 12 days, the increase was 3.7-fold (p < 0.01), and in 26-day-old animals it was 14.9-fold (p < 0.001). However, in adult rats in random stages of the estrous cycle, the variation was wide and TABLE 2. Effects of hCG treatment (600 lU/kg B.W., single injection) on body and ovarian weights 3 days after the treatment. Age (days) 8 9 10 11 12 13 26 Adults

Weights (g) hCG treated Controls 14.61 19.13 18.70 19.17 21.51 25.67 61.40 289.8

-+0.39 + 0.31 + 0.55 + 0.29 + 0.85 - 0.55 + 2.37 + 17.83

15.43 + 0.37 17.49 + 0.66 18.67 + 0.46 20.02 + 0.57 + 0.46 21.82 26.93 + 0.68 65.20 + 3.51 289.4 + 11.49

2

Ovarian weights (mg) hCG treated Controls 0.41 0.67 0.80 0.82 0.90 1.21 6.27 39.6

+ 0.03 + 0.05 + 0.05 + 0.05 + 0.07 + 0.09 + 0.60 + 3.40

0.47 + 0.04 0.49 + 0.04* 0.96 + 0.06 0.98 + 0.05* 1.22 + 0.15 2.13 + 0.10*** 11.73 + 0.42*** 68.90 + 8.50**

'Control and hCG treatment groups consisted of 5-18 animals (mean + SEM). 2 *p < 0.05, **p < 0.01, ***p < 0.001 vs. controls.

the difference was not statistically significant. Basal P levels remained low in the group aged 8 to 13 days (< 87 fmol/ ovary). Treatment with hCG increased the ovarian T contents for the first time at the age of 11 days, on Day 3 after injection of hCG. The hCG-stimulated increase in T production was highest, 47-fold, at the age of 13 days (p < 0.001, Fig. 2d). Consistent elevation in ovarian weights after hCG treatment was observed after 13 days of age (Table 2). There were no changes in total body weights of any of the treatment groups (Table 2). Combined FSH and hCG Treatment In rats pretreated with FSH during Days 3-8 (0.3 IU recFSH twice daily) and injected with hCG at the age of 6 days, the suppression of serum LH levels was significant (p < 0.01) only in the group receiving the combined FSH + hCG treatment (Fig. 3a). The ovarian contents of E2 did not differ between different treatment groups (Fig. 3b). Only the single injection of hCG increased the ovarian P contents, about 1.8-fold (p < 0.001, Fig. 3c). A significant increase in ovarian T was again observed only in the combined FSH + hCG treatment group (p < 0.05, Fig. 3d).

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MATURATION OF THE PITUITARY-GONADAL AXIS

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AJ" 50-fold compared with the NIDDK RIA reagents), small sample volumes from neonatal rats can be analyzed reliably. The basal levels of serum LH in groups aged 5-12 days were about 3-fold higher than those in adults, and the biological variation was wide, in accordance with previous investigations [12, 28, 29]. Serum LH was significantly suppressed for the first time in neonatal female rats at the age of 10 days as measured 3 days after a single injection of hCG. This finding coincides with the appearance of the full-length mRNA [10] and the functional protein for the LH receptor in the rat ovary at the age of 7 days [4-8]. The hCG-stimulated steroidogenesis must apparently persist for 3 days before the suppression of LH can be seen. This was the case in the 7-10-day group, but not in the 6-

9-day treatment group. Likewise, the suppressive effect'of hCG on LH secretion was not achieved in neonatal rats treated at the age of 7 days and killed one day later, despite significant increases in intraovarian E2 and T contents. When these steroids were measured on Day 10, three days after the hCG injection, the increase in steroids was no longer significant. However, the overall stimulation of steroidogenesis in these animals must have been significant, since serum LH was suppressed. In the time course study with 10-13-day-old rats, hCG had clear effects on steroidogenesis: intraovarian T increased 60-fold and E2 increased 22-fold on Day 2 after the hCG injection. A more gradual, 10-fold increase occurred in P. Despite the acute effects on steroidogenesis, the suppressive effect of hCG on LH secretion was achieved only on Day 3 after the hCG injection. As discussed above, the stimulation of steroidogenesis apparently must last for several days in the immature animal before the pituitary is suppressed. Although the ovarian content of E2 was significantly increased by hCG by Day 11, the high binding of E2 to serum ot-fetoprotein [14,17] in neonatal female rats up to the age of 25 days indicates that the role of E2 in the feedback regulation is uncertain. The marked T response indicates that

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SOKKA AND HUHTANIEMI 70 -

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FIG. 3. Response of serum LH concentrations (a) and intraovarian contents of E2 (b), P (c), and T (d) to FSH treatment (0.3 IU recFSH, twice daily, on Days 3-8 of life) or combined FSH treatment (vs. above) and hCG stimulation (600 IU/kg BW, single injection) on Day 6. The values are mean + SEM of measurements from 10-18 animals. Different letters above the bars indicate a statistically significant difference between the groups (p at least < 0.05) (MannWhitney U-test for serum LH, one-way ANOVA and Duncan's New Multiple Range test after logarithmic transformation for ovarian steroids).

ovarian steroids other than E2 can be responsible for the early negative feedback on LH secretion. E2 has been reported to replace androgens in the negative feedback after Day 15 of life, a change that coincides with the disappearance of ot-fetoprotein from the circulation [28]. The data on treatment of neonatal rats with diethylstilbestrol, which does not bind to -fetoprotein [17], indicate the effectiveness of this synthetic estrogen in suppressing circulating LH levels after ovariectomy as early as on Day 10 of life [16]. Treatment with hCG increased ovarian T content significantly after Day 11, and, if measured one day after stimulation, as soon as Day 8. Concentration of T was greatest at 13 days of age and declined by 26 days of age. The increased T content after hCG treatment is in harmony with previous reports suggesting that androgens have a major role in the negative feedback regulation of gonadotropins in infantile rats [15, 30]. However, the mechanism is probably not very effective, or the androgen concentrations are too low, because the basal serum gonadotropin levels at this age are elevated. The role of androgens in the development of the neonatal ovary is not clear, but T may contribute to the timing of vaginal opening [31]. Alternatively, the effect of androgens on negative feedback regulation can also be explained by local aromatization to estrogens in the hypothalamus or pituitary. This could be supported by the finding that 5(x-reductase activity is elevated in the anterior

pituitary of neonatal rats, reaching a maximum on Day 12 [32]. We also have to consider the possibility, although it is unlikely, that hCG has direct effects on LH biosynthesis and secretion through direct effects at the level of the hypothalamus or the anterior pituitary [33-35]. Studies have demonstrated the existence of a rapid short-loop feedback system for the hypothalamic-pituitary control of LH secretion. The possibility of extragonadal actions of gonadotropins is further substantiated by the recent discovery of ubiquitous extragonadal LH receptor gene expression, including expression in the central nervous system [36, 37]. However, before the physiological role of the extragonadal LH receptors is established, we can conclude that the appearance of LH receptors in the ovary at the age of 7 days postpartum is the last link in the appearance of the pituitary-gonadal regulation circuit. As soon as this occurs, LH/ hCG is able to stimulate the ovarian steroidogenic response that subsequently inhibits LH secretion. FSH is known to induce LH receptors in the adult ovary [18,19]. By treating the animals from Day 3 onwards with recFSH, we wanted to test whether increased FSH advances the postnatal appearance of the ovarian LH response. Such an effect appeared possible, since the FSH receptor mRNA [9] and FSH receptor protein [3, 4] are present in postnatal ovary on Days 1 and 4, respectively. Combined treatment with FSH and hCG suggested that elevated FSH levels ad-

MATURATION OF THE PITUITARY-GONADAL AXIS

vance the onset of hCG-stimulated steroidogenesis and pituitary feedback responses. This finding corroborates a previous study in which treatment of neonatal female mice with equine chorionic gonadotropin stimulated follicular growth

[381.

In conclusion, the negative feedback effect of the hCGstimulated neonatal ovary on pituitary LH secretion is seen simultaneously with the appearance of functional LH receptors. However, several days of hCG stimulation are needed at this age before the pituitary responds with suppressed LH secretion. The functional LH receptor is apparently the last link to appear in the hypothalamic-pituitaryovarian regulatory circuit. The concomitant stimulation of ovarian steroidogenesis in response to exogenous hCG suggests that ovarian steroids, most likely androgens, initially mediate the negative feedback response. Elevated postnatal levels of FSH do advance the appearance of the ovarian LH response as monitored by the timing of stimulation of steroidogenesis and the onset of negative feedback of gonadal hormones on LH secretion. ACKNOWLEDGMENTS The authors wish to thank Ms. A. Metsvuori and Ms. T. Laiho for excellent technical assistance.

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