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I. HUHTANIEMI

SUMMARY

The neutral steroids derived from their conjugates present in a pool of bile from 20 human foetuses were analysed by gas—liquid chromatography and gas chromatography— mass spectrometry. Ten monosulphates, twelve disulphates and seven glucuronides were detected, but no free steroids were found. Both saturated and unsaturated steroids of the C19- and C21-series were detected. The glucuronides of 5β-pregnane-3α,20α-diol, 5α-pregnane-3β,16α,20α-triol and 3α-hydroxy-5β-pregnan-20-one as well as the monosulphate of 16α-hydroxydehydroepiandrosterone were present in the highest concentrations. Other major components were the monosulphates of dehydroepiandrosterone, pregnenolone and 16α-hydroxypregnenolone and the disulphates of 5-androstene-3β,17α-diol, 3β, 17β-dihydroxy-5-androsten-16-one and 5β-prenane-3ξ,20α,21-triol. The total concentration of the monosulphate conjugates was 435 μg/100 g sample (wet weight), of the disulphates 363 μg/100 g and of the glucuronides 815 μg/100 g.

Many of the compounds detected have previously been found in the foetal liver and intestinal contents. It is therefore concluded that during early and mid-gestation the foetal liver is already excreting many steroid conjugates through the biliary tract into the intestinal canal. As in other foetal compartments, considerable amounts of steroid sulphates were detected. However, the presence of steroid glucuronides in the bile suggests that the formation of hepatic glucuronides already occurs during the foetal period.

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M Bergendahl and I Huhtaniemi

Abstract

Short-term starvation suppresses the pituitary-testicular function in rats, evidently through inhibition of gonadotrophin-releasing hormone (GnRH) release. However, when gonadotrophin secretion is strongly enhanced, e.g. after castration, starvation does not suppress gonadotrophins. To test whether the time since castration affects the pituitary response to starvation, adult male rats were totally deprived of food for five days (only water allowed) immediately (acute castration) or two weeks after castration (chronic castration). The pituitary contents of GnRH receptors were decreased by starvation in sham-operated animals, unaffected in acutely castrated rats, but increased in chronically castrated animals, in comparison with appropriate controls (P<0·01). Castration per se increased steady-state mRNA levels of the common α-chain and the LH and FSH β-chains in all groups studied. The only consistent effect of starvation on these parameters was the 1·7 to 2-fold increase in the pituitary content of LH β-subunit mRNA in acutely and chronically castrated rats (P<0·01). Starvation alone suppressed LH secretion, acute castration eliminated this effect, but in chronically castrated rats, the starvation effect was stimulatory. Starvation did not affect FSH secretion in sham-operated and acutely castrated rats, but after chronic castration, the effect was stimulatory. In conclusion, the overall effect of starvation on gonadotrophins shifts gradually after castration from suppression, in sham-operated rats, to stimulation, in chronically castrated animals. Parallel changes in pituitary GnRH receptors suggest similar changes in GnRH secretion. Hence, starvation has both negative and positive effects on the GnRH-gonadotrophin-axis. The negative effect is evidently androgen-dependent and dominates in testes-intact animals. After chronic castration, only the positive, non-androgen dependent, stimulatory effect remains.

Journal of Endocrinology (1994) 143, 209–219

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I. HUHTANIEMI and R. VIHKO

SUMMARY

Sulphate-conjugated neutral steroids in the intestinal contents of early and mid-term human foetuses (14–20 weeks of gestation) were investigated by gas—liquid chromatography and gas chromatography—mass spectrometry. Twenty-four neutral steroids were found in the monosulphate fraction and 21 in the disulphate fraction. The total concentration of these steroids varied between 9·6 and 15·3 mg/100 g meconium wet weight, about half being monosulphates and half disulphates. Steroids with a 3β-hydroxy-5-ene structure were found and also saturated steroids which carried hydroxyl groups at carbons 3, 11, 15, 16, 17, 18, 19, 20 or 21. Among them were steroids not previously detected in human foetal compartments. Thus, the foetal meconium is both qualitatively and quantitatively the richest foetal source of neutral steroid sulphates so far investigated.

The present results together with those reported in the literature show that in the course of pregnancy there is a many-fold increase in the steroid concentration of the foetal intestinal contents. This is most obvious in the case of saturated C19 steroids and polar pregnane derivatives. Reduction of the 20-ones to 20β-ols seems to be more extensive towards the end of pregnancy. 5, 16-Pregnadienes and 3β-hydroxy-5β-pregnanes are present in higher concentrations in early pregnancy.

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P. Pakarinen and I. Huhtaniemi

ABSTRACT

The responses of gonadotrophin gene expression, pituitary content and serum levels to castration alone and castration plus testosterone replacement (silicone elastomer implants) were compared in male rats at 10, 30, 60 and 90 days of age. Sham-operated animals served as controls. In addition, 30-day-old castrated rats were treated with dihydrotestosterone (DHT) and diethylstilboestrol (DES). When killed 7 days after castration, the increases in serum LH (six- to eightfold; P < 0·01) and FSH (two- to fourfold; P < 0·01) were similar at all ages studied. Likewise, testosterone reversed the effects of castration in a largely similar fashion at all ages. In contrast, great age-related differences were observed in the responses of gonadotrophin subunit mRNAs to the treatments. Castration increased the common α subunit mRNA two- to fourfold on days 10 and 30 (P < 0·01), sixfold on day 60 (P < 0·01), but not at all on day 90. Testosterone reversed the increases at all ages, but the levels were below those of controls only at 90 days (P < 0·01). The highest increases (sixfold; P < 0·01) of LH-β mRNA were seen on days 10 and 60, the others being two- to threefold higher (P < 0·05–0·01). Testosterone reversed this effect at 60 days and suppressed LH-β mRNA to below the control levels at other ages (P < 0·01). Castration had no effect on FSH-β subunit mRNA at 30 and 90 days but a four-to fivefold increase was seen on days 10 and 60 (P < 0·01). Testosterone suppressed these mRNAs at all ages, and they decreased to below the levels in controls at 30 and 60 days. Testosterone, DHT and DES had, at 30 days, practically the same effects on the LH parameters, whereas DHT was clearly less effective than testosterone and DES in suppressing those of FSH. In conclusion, although there was, in general, good agreement between gonadotrophin mRNA and serum levels in response to castration and testosterone replacement, there were specific ages when the post-castration increases in FSH and/or LH occurred with no detectable change in the respective mRNA levels. These findings indicate that altered transcription (or mRNA stability) is not solely responsible for the responses of the gonadotrophins to altered gonadal feedback, but that changes in translation efficiency and/or serum gonadotrophin stability are involved at specific ages of development.

Journal of Endocrinology (1992) 135, 507–515

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T. Sokka and I. Huhtaniemi

ABSTRACT

The sequence of appearance of FSH and LH receptors, and response of cyclic AMP (cAMP) production to these hormones and cholera toxin, were studied in the fetal and neonatal rat ovary. Specific binding of radio-labelled human (h)FSH and chorionic gonadotrophin (CG) to ovarian homogenates was first detectable on day 7 of life. The content of FSH receptors per ovary increased tenfold between days 7 and 16, and that of LH receptors 27-fold. A significant response of cAMP production in vitro to FSH appeared on day 4 of life, but no significant effect of hCG on cAMP was achieved until day 7. In contrast, cholera toxin had a marked effect on cAMP production by day 17 of fetal life. Although both FSH and LH receptors were detectable in the neonatal rat ovary by day 7, the present findings indicate that the FSH responsiveness of the ovary appears earlier than that of LH. The post-receptor machinery of cAMP production is already functional in the fetal ovary as shown by the experiments with cholera toxin. The appearance of the receptor may therefore be the last link in the ontogeny of the gonadotrophin signal transduction system in the ovary. To study the hormone dependence of the appearance of gonadotrophin responsiveness, neonatal female rats were treated on days 1–6 or 1–9 of life with a potent gonadotrophin-releasing hormone antagonist, and killed on the following day. In both treatment groups, the pituitary LH and FSH contents were suppressed. The body weights remained unaltered, but ovarian weights decreased significantly during both periods of treatment (days 1–6,26·1%, P < 0·05; days 1–9,54·0%, P <0·001). No difference in basal or FSH-stimulated cAMP production was achieved by antagonist treatment for the first 6 days of life. The basal and hCG-stimulated rates of cAMP production per ovary were reduced in animals treated for 9 days (P <0·01), but the FSH-stimulated cAMP production remained unaffected. Hence, whereas the responsiveness to FSH seems to develop in the absence of normal gonadotrophin secretion, a causal relationship between normal gonadotrophin levels and the appearance of LH/hCG responsiveness is apparent in the neonatal rat ovary.

Journal of Endocrinology (1990) 127, 297–303

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K.-L. Kolho and I. Huhtaniemi

ABSTRACT

The acute and long-term effects of pituitary-testis suppression with a gonadotrophin-releasing hormone (GnRH) agonist, d-Ser(But)6des-Gly10-GnRH N-ethylamide (buserelin; 0·02, 0·1, 1·0 or 10 mg/kg body weight per day s.c.) or antagonist, N-Ac-d-Nal(2)1,d-p-Cl-Phe2,d-Trp3,d-hArg(Et2)6,d-Ala10-GnRH (RS 68439; 2 mg/kg body weight per day s.c.) were studied in male rats treated on days 1–15 of life. The animals were killed on day 16 (acute effects) or as adults (130–160 days; long-term effects). Acutely, the lowest dose of the agonist decreased pituitary FSH content and testicular LH receptors, but with increasing doses pituitary and serum LH concentrations, intratesticular testosterone content and weights of testes were also suppressed (P< 0·05–0·01). No decrease was found in serum FSH or in weights of accessory sex organs even with the highest dose of the agonist, the latter finding indicating continuing secretion of androgens. The GnRH antagonist treatment suppressed pituitary LH and FSH contents and serum LH (P< 0·05–0·01) but, as with the agonist, serum FSH remained unaltered. Testicular testosterone and testis weights were decreased (P <0·01) but testicular LH receptors remained unchanged. Moreover, the seminal vesicle and ventral prostate weights were reduced, in contrast to the effects of the agonists.

Pituitary LH and FSH contents had recovered in all adult rats treated neonatally with agonist and there was no effect on serum LH and testosterone concentrations or on fertility. In contrast, in adult rats treated neonatally with antagonist, weights of testis and accessory sex organs remained decreased (P <0·01–0·05) but hormone secretion from the pituitary and testis had returned to normal except that serum FSH was increased by 80% (P <0·01). Interestingly, 90% of the antagonist-treated animals were infertile.

It is concluded that treatment with a GnRH agonist during the neonatal period does not have a chronic effect on pituitary-gonadal function. In contrast, GnRH antagonist treatment neonatally permanently inhibits the development of the testis and accessory sex organs and results in infertility. Interestingly, despite the decline of pituitary FSH neonatally, neither of the GnRH analogues was able to suppress serum FSH values and this differs from the concomitant changes in LH and from the effects of similar treatments in adult rats.

Journal of Endocrinology (1989) 123, 83–91

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K.-L. Kolho and I. Huhtaniemi

ABSTRACT

Suppression of neonatal rat pituitary-testis function by gonadotrophin-releasing hormone (GnRH) antagonists results in delayed sexual maturation and infertility. Since the mechanism is not understood, the acute effects of a GnRH antagonist on gonadotrophin secretion in neonatal male rats has been studied in more detail. Treatment with a GnRH antagonist analogue, N-Ac-d-Nal(2)1,d-p-Cl-Phe2,d-Trp3,d-hArg(ET2)6,d-Ala10-GnRH (2 mg/kg per day) on days 1–10 of life had prolonged effects on gonadotrophin secretion; serum LH and FSH recovered in 1 week, but the pituitary content took 2 weeks to recover. Likewise, LH and FSH responses to acute in-vivo stimulation with a GnRH agonist were still suppressed 1 week after the treatment. Interestingly, a rebound (86% increase) in basal serum FSH was found 16 days after treatment with the antagonist.

Whether testis factors influence gonadotrophin secretion during treatment with the GnRH antagonist and/or in the subsequent recovery period was also assessed. Neonatal rats were castrated on days 1, 5 or 10 of the 10-day period of antagonist treatment. Orchidectomy on days 1 and 5 only marginally affected gonadotrophin secretion. When orchidectomy was performed at the beginning of the recovery period, no effects on pituitary recovery were seen within 1 week of castration. After 16 days, serum LH and FSH in the antagonist-treated and control castrated rats were equally increased but the pituitary contents of the antagonist-treated rats were still suppressed.

Finally, the effect of testosterone treatment on the recovery of gonadotrophin secretion after antagonist suppression was studied in intact and orchidectomized animals. The rats were implanted with testosterone capsules for 7 days after treatment with the GnRH antagonist in the neonatal period. Testosterone suppressed pituitary LH contents similarly in all groups of animals, but had no effects on serum LH. Paradoxically, serum FSH was suppressed 50% by testosterone in intact and castrated antagonist-treated rats and in castrated controls but not in intact controls. These findings suggest that suppression of FSH by testosterone is only seen in neonatal animals with low endogenous levels of this androgen, whether due to GnRH antagonist treatment or castration.

It is concluded that neonatal treatment with a GnRH antagonist results in prolonged suppression of LH and FSH secretion, that testis factors play only a minor role in pituitary modulation during the antagonist suppression and that more disturbances are observed in the post-treatment recovery of FSH secretion than in that of LH.

Journal of Endocrinology (1989) 122, 519–526

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Rilianawati, J Kero, T Paukku and I Huhtaniemi

We have developed a transgenic (TG) mouse model for tumorigenesis of gonadal somatic cells using a 6 kb fragment of the mouse inhibin-alpha subunit promoter (Inh-alpha) fused with the simian virus 40 T-antigen (Tag) coding sequence. Gonadal tumors, of Leydig or granulosa cell origin, develop in the TG mice with 100% penetrance by the age of 5-8 months. Conspicuously, if the mice are gonadectomized, they develop adrenal tumors. Gonadal and adrenal tumorigenesis in these mice seem to be gonadotropin dependent. On the other hand, testosterone stimulates the proliferation of a cell line (C alpha 1) established from one of the adrenal tumors. The purpose of the present study was therefore to investigate further whether testosterone affects the growth of these gonadal and adrenal tumors in vivo. Two experimental models were used: (1) Tag TG/hypogonadotropic (hpg) double mutant mice and (2) castrated Tag TG mice. Both were treated between 1-2 and 7-8 months of age with Silastic rods (length 2 cm) containing testosterone. None of the control or testosterone-treated Tag/hpg mice developed gonadal or adrenal tumors. The castrated Tag TG mice displayed, upon microscopical examination, early stages of adrenal tumors, whereas those receiving testosterone did not show such changes. Testosterone increased the weights of gonads in the Tag/hpg mice, and those of uteri and seminal vesicles in both groups. In contrast, the adrenal weights were significantly reduced in both groups by testosterone treatment. Gonadal histology of the testosterone-treated mice showed hyperplasia of testicular Leydig cells and ovarian stroma. Spermatogenesis was induced by testosterone in the Tag/hpg mice. Adrenal histology of the testosterone-treated animals demonstrated the disappearance of the X-zone. Serum levels of FSH in testosterone-treated Tag/hpg mice were significantly increased, while those of serum LH were decreased. In conclusion, the present result indicate that the suppression of gonadotropins by testosterone implants in castrated Inh-alpha/Tag TG mice prevents the tumorigenesis of their adrenals. In intact Tag/hpg mice, testosterone implants were not able to induce gonadal or adrenal tumorigenesis. Although testosterone treatment was able to induce interstitial cell hyperplasia in gonads of the Inh-alpha/Tag mice, direct gonadotropin action is responsible for gonadal and adrenal tumorigenesis.

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K.-L. Kolho, H. Nikula and I. Huhtaniemi

ABSTRACT

Postnatal secretion of gonadotrophin by male rats was inhibited by a potent gonadotrophin-releasing hormone (GnRH) antagonist analogue (N-Ac-4-Cl-d-Phe1,4-Cl-d-Phe2,d-Trp3,d-Phe6,des-Gly10-GnRH-d-alanylamide; Org 30039; 2 mg/kg s.c. twice daily) on days 1–5, 6–10, 11–15 or 16–20 of life. The onset of puberty was determined by monitoring the separation of the preputium from the glans penis, i.e. balanopreputial separation (BPS). Rats treated on days 1–5 matured normally, whereas all treatments between days 6 and 20 delayed BPS (P < 0·01). In adult rats (between 110 and 160 days of age), testis weights were reduced by 21–35% (P < 0·01) in groups treated between days 1 and 15, although weights of the accessory sex glands were normal. Testicular FSH receptors were decreased by 31–47% (P < 0·01) in all treatment groups, whereas the LH receptor content was decreased only in rats treated between days 1 and 5 (18%; P < 0·05) and prolactin receptor content decreased only in rats treated up to day 10 (31–33%; P < 0·01). Concentrations of serum testosterone, LH and FSH, and pituitary contents of LH and FSH were unaffected by neonatal treatment with Org 30039. Animals treated with Org 30039 had reduced fertility which was most pronounced (88%; P < 0·01) in rats treated between days 1 and 5. However, motile sperm were detectable in the cauda epididymis of the infertile rats.

In conclusion, postnatal gonadotrophin deprivation induced with a GnRH antagonist for different 5-day periods during the first 15 days of life delayed puberty, reduced adult testis weight and impaired fertility. Some effects of the antagonist were largely independent of the timing of gonadotrophin suppression. Other effects, including suppression of testicular LH and prolactin receptors and the delay in the onset of puberty, were found only in the younger and older treatment groups respectively. These findings emphasize the importance of neonatal hypothalamic-pituitary-gonadal function for subsequent sexual maturation.

J. Endocr. (1988) 116, 241–246

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BP Setchell, P Pakarinen and I Huhtaniemi

The purpose of this study was to assess the concentrations of LH that Leydig cells are exposed to upon in vivo stimulation of steroidogenesis. The concentrations of LH were measured in rats in testicular interstitial extracellular fluid, seminiferous tubular fluid and blood plasma from testicular veins from one testis before and from the other testis of the same rats after an intravenous injection of gonadotrophin-releasing hormone (GnRH) or saline, and compared with the concentrations in blood plasma from a peripheral vein. The concentrations of LH in interstitial fluid surrounding the Leydig cells before the injections were about 10% of the levels in blood plasma, and showed no significant rise at 15 min and a much smaller rise at later times in rats injected with GnRH than those seen in blood plasma from either of the two sources, which were similar. The concentrations of LH in tubular fluid were even lower and showed no change after GnRH. Testosterone concentrations in testicular cells, interstitial fluid and testicular venous blood plasma were significantly increased by 15 min after GnRH, when compared with saline-injected controls, with no change in the levels in tubular fluid. The rise in testosterone concentrations in testicular venous plasma after GnRH was smaller than those in the cells and interstitial fluid. In conclusion, the concentrations of LH reaching the testicular interstitial fluid were only about one-tenth of that measured in the circulation, presumably because the endothelial cells restrict access of the hormone to the interstitial fluid. This indicated that either the Leydig cells are extremely sensitive to LH stimulation or that testicular endothelial cells modulate the action of LH on the Leydig cells.