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Specific high-affinity low-capacity binding of human chorionic gonadotropin (hCG) and lutropin (hLH) was demonstrated in the plasma membrane fractions of human corpora lutea. The number of binding sites for both hormones increased from early to late luteal phase, whereas regressing corpus luteum from proliferative phase did not bind either hormone. On the basis of apparent dissociation constants the affinity of the receptor for hLH is highest during early luteal phase and decreases toward the end of the cycle, which may reflect an increasing insensitivity of the corpus luteum to circulating hLH. By contrast, the affinity of the receptor for hCG is highest in the midluteal phase. There are gonadotropin binding sites in the human corpus luteum also during pregnancy, but they are saturated by endogenous hCG. Evidence for this was obtained by elution of hCG with 0.15M sodium chloride at pH 2.3 from washed plasma membrane fractions of luteal tissue from six to 16 week's gestation. After acid treatment and neutralization these preparations showed specific binding for 125I-labeled hCG, but not for 125I-labeled hLH. Our results demonstrate a shift in the balance of affinity of the gonadotropin receptor from hLH to hCG during the course of luteal phase, and during pregnancy the binding sites appear to be available for hCG only.

The histochemical localization of four dehydrogenase systems (diphosphopyridine nucleotide diaphorase, lactic, succinic and steroid-3β-ol-dehydrogenase) has been studied in human ovary during the menstrual cycle. The phase of the cycle was determined using endometrial biopsy and vaginal smear examination and by estimations of the urinary excretion of pregnanediol-complex. All the dehydrogenases were found to exhibit equal distribution; the most active structures were the theca interna cells of the developing follicles and both thecal and granulosa lutein cells of the corpora lutea. Atretic follicles as well as corpora albicans consistently showed moderate dehydrogenase activity. Formazan deposit was always developed in the interstitial cells of the stroma. No changes were detected in the enzyme activity during the follicular and progestational phases of the cycle.

Free and sulfate-conjugated neutral steroids were isolated from the pooled testes of 33 human fetuses of 8.5 – 20.0 cm crown-rump length and the compounds were identified by gas-liquid chromatography and gas chromatography — mass spectrometry. Unconjugated pregnenolone and testosterone were the main compounds present. Thus it has been established that under in , vivo conditions the fetal testis synthesizes androgens and that apparently the pathway pregnenolone → 17a-hydroxypregnenolone → dehydroepi-androsterone → androstenedione/5-androstene-3β, 17β-diol → testosterone is preferred.

The ovaries of ten patients, at least one year after the post-menopause, were incubated with two Δ5-C19-steroids and also studied histochemically. All these patients had post-menopausal uterine bleeding and increased oestrogen excretion of the urine. The urinary estimations of gonadotrophins, 17-KS, 17-OHCS and pregnanediol were carried out on all patients. Vaginal smears were read according to Papanicolaou, and the endometrium and ovaries were studied histologically. The incubation experiments indicate the presence of Δ5-3β-hydroxysteroid-dehydrogenase. When androst-5-ene-3β,17β-diol was used as precursor the formation of testosterone occurred without any concomitant production of DHA and/or androstenedione. This seems to indicate the possible role of the Δ5-pathway in the formation of testosterone by post-menopausal ovarian tissue. The histochemical reactions indicated a reducing activity on NADH, lactate and glucose-6-phosphate, in certain corpora albicantia, atretic follicles and in diffuse thecoma regions in the cortical layer of the ovary. Steroid-3β-ol-dehydrogenase and β-hydroxybutyrate-dehydrogenase were found only at the edges of certain corpora albicantia, in some individual stroma cell groups and in some atretic follicles. Our studies, both biochemical and histochemical, suggest that the observed increase in the urinary oestrogens of the patients studied might in part at least, be of ovarian origin. This opinion is also supported by the postoperative oestrogen values.

Sections of testes from nine human foetuses ranging in crown-rump length from 3.0 to 18.3 cm were incubated to determine 3β-hydroxy-steroid dehydrogenase activity histochemically with the following steroids: 3β-hydroxy-pregn-5-en-20-one (pregnenolong). 3β,17α-dihydroxy-pregn-5-en-20-one (17α-hydroxypregnenolone). 3β-hydroxy-androst-5-en-17-one (DHA). 3β,17β-dihydroxy-androst-5-ene (androstenediol). 3β-sulphoxy-pregn-5-en-20-one (pregnenolone sulphate). 3β-sulphoxy-1 7α-hydroxy-pregn-5-en-20-one (17α-hydroxy-pregnenolone sulphate) 3β-sulphoxy-androst-5-en-17-one (DHAsulphate). 3β-hydroxy-5α-androstan-17-one (epiandrosterone). Pregnenolone and DHA gave a colour reaction in the interstitium of all testes studied. 17α-hydroxypregnenolone was utilised by testes from foetuses of C-R length 8.8 cm and over, androstenediol by testes from foetuses of C-R length 6.1 cm and over. These facts are thought to support the concept of separate substrate-specific 3β-hydroxysteroid dehydrogenases in the testis. Pregnenolone sulphate was used by the interstitial cells of all testes studied but gave a stronger reaction than the free steroid. 17α-hydroxy-pregnenolone sulphate was used by all foetal testes surveyed. DHA sulphate was not well used by the interstitial cells. The utilisation of steroid sulphates in a different manner from the free steroids in this histochemical system may mean that the presence of a sulphate group affects enzyme-substrate binding or that a steroid sulphatase is involved. Intense formazan deposition followed incubation with epiandrosterone in all testes studied. This seems to imply that a δ5 configuration is not necessary for enzyme-substrate binding.

THERE is a considerable quantity of evidence demonstrating that the fœtal testis produces a hormone responsible, for example, for the freemartin effect1. Explanation of fœtal rat testes has shown that secretion of the hormone begins when the fœtus is 15–16 days old2,3. Morphological differentiation of the Leydig cells begins during the same period, the interstitial spaces being filled by closely packed polygonal cells containing periodic-acid–Schiff-positive material4.

The histochemistry of 4 enzyme systems (nonspecific esterase, DPNH, β-hydroxybutyrate and steroid-3β-ol dehydrogenases) and. of lipids has been studied in the Leydig tissue of the rat from the 3rd to the 50th postnatal day. The distribution and form of the Leydig cells as revealed by these reactions were different in the prepubertal period as compared with maturity. In both, the Leydig tissue exhibited all the enzyme activities. The number of positive cells diminished rapidly during the 1st postnatal week but more slowly during the 2nd and 3rd weeks. At puberty, enzymatically active Leydig cells appeared evenly distributed in the intertubular tissue. Prepubertal Leydig cells contained sudanophilic lipid material, whereas those of the mature animal did not exhibit any stainable lipids. On the basis of these results and some earlier observations, it is concluded that there are 2 different generations of Leydig cells in the testis of the rat.

THE biosynthetic pathways of androgens in the adult mammalian testis have recently been fully described1. The pathways include the formation of testosterone from pregnenolone through progesterone, 17α-hydroxyprogesterone2, 17α-hydroxypregnenolone and dehydroepiandrosterone3. Although androstenedione was thought to be the common precursor for testosterone in both pathways, the importance of androstenediol as an alternative intermediate step in the latter pathway has been seen in the rabbit testis4.

ALTHOUGH various functions have been suggested for the pineal gland1, very few positive findings have been made concerning this gland2,3. In this communication histochemical evidence is given of a strong enzyme activity in the rat's pineal gland capable of splitting L-leucyl-β-naphthylamide hydrochloride. Recent studies4,5 indicate that this activity is actually due to a group of peptidases, including ‘leucine aminopeptidase’.