Henk J. Van Der Molen

The role of glucose, pyruvate and lactate in ATP production by rat spermatocytes and spermatids

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous L-lactate (3-6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.

Comparison of the effects of insulin and follitropin on glucose metabolism by Sertoli cells from immature rats.

Sertoli cells were isolated from the testes of 3-week-old sterile rats (prenatally irradiated) and incubated for 3 days in the absence of added hormones. Subsequently the effects of follitropin and insulin on glucose metabolism were investigated using this in vitro system. A marked stimulation of net lactate production by either follitropin or insulin was observed within 3 h after addition of the hormones. This response was not inhibited in the presence of the protein synthesis inhibitor cycloheximide. Production of cAMP by the Sertoli cells was markedly enhanced by follitropin, but not at all by insulin. The addition of 0.5 mM dibutyryl cAMP to the incubation medium also resulted in a rapid increase of the rate of lactate production by the Sertoli cells. The stimulation of lactate production by follitropin and insulin was dose-dependent (ED50 of approx. 10 ng NIH-FSH-S13/ml and of approx. 50 ng insulin/ml). It is suggested that the observed effects of insulin on Sertoli cells are mediated via insulin receptors, rather than via receptors for insulin-like growth factors. Within 18 h after addition of either follitropin or insulin the cells became refractory with respect to lactate production to the homologous hormone, whereas the cells could still respond to the heterologous hormone. It is concluded, that follitropin and insulin, acting via different mechanisms, exert similar rapid stimulatory effects on glucose metabolism by Sertoli cells from immature rats in vitro. These effects are not dependent on de novo protein synthesis and may differ from long-term trophic effects of follitropin, insulin, and/or insulin-like growth factors.

Hormonal regulation of lh stimulation of testosterone production in isolated leydig cells of immature rats: The effect of hypophysectomy, fsh, and estradiol-17β

Testosterone production in isolated Leydig cells from testes of immature and adult rats was stimulated by addition of LH in a dose dependent way. Hypophysectomy of adult rats had no influence on LH-stimulated testosterone production in isolated Leydig cells after 5 days. In contrast hypophysectomy of immature rats resulted after 5 days in an almost complete loss of LH sensitivity of isolated Leydig cells. Daily adminitration of FSH during 5 days starting immediately after hypophysectomy maintained LH responsiveness of isolated Leydig cells of immature rats. Also FSH administration starting on day 5 after hypophysectomy resulted in a restoration of LH responsiveness. Estradiol benzoate, injected simultaneously with FSH, abolished the FSH-induced LH responsiveness.

Characterization of a nuclear receptor for testosterone in seminiferous tubules of mature rat testes.

Abstract A specific androgen receptor could be demonstrated in the nuclear and cytoplasmic fractions of testicular tissue of mature hypophysectomized rats, either in vivo after injection of testosterone or in vitro after incubation of testis tissue with testosterone. Using agar-gel electrophoresis this receptor could be distinguished from the testicular transport-like protein for androgens (androgen binding protein = ABP). After in vivo administration of testosterone the steroid bound to the receptor in mature rat testis was mainly unmetabolized testosterone. After dissection of testis tissue the larger part of the receptor was shown to be present in the seminiferous tubules. The amount of exogenous testosterone that could be bound per mg of protein in the nuclear extract increased gradually during 20 days after hypophysectomy. Some characteristics of the receptor in the nuclear extract and of ABP were compared : the receptor was more sensitive to temperature increases than ABP; the steroid dissociated more slowly from the receptor than from ABP; cyproterone acetate showed almost no effect on the binding of dihydrotestosterone to ABP, but did compete for the receptor binding sites in the nuclear extract.

A receptor for testosterone in mature rat testes

In most steroid target tissues the steroids can be bound by specific receptors in the cytosol and in the nuclear fraction. Androgens are known to affect spermatogenesis [l] , it was therefore of interest to investigate if specific androgen receptors are present in testis tissue. In this paper we discuss results obtained for the binding of testosterone in nuclear fractions of seminiferous tubules of mature hypophysectomized rats.

The possible role of protein kinase C and phospholipids in the regulation of steroid production in rat Leydig cells

We have studied the possible involvement of the activation of calcium‐dependent phospholipid‐activated protein kinase (PK‐C) in the stimulatory action of LHRH on Leydig cells, using 4β‐phorbol‐12‐myristate‐13‐acetate (PMA) and phospholipase C (PL‐C). LHRH agonist (LHRH‐A) and PL‐C had a large synergistic effect on LH‐stimulated steroid production, whereas PMA inhibited the effect of LH. However, PMA always caused an increase in steroid production stimulated by various doses of dibutyryl cAMP. LH and PMA stimulated the phosphorylation of 17 and 33 kDa proteins, whereas LHRH‐A and PL‐C had no effect. Of all effectors used, LH had the most pronounced effect on the synthesis of 14, 27 and 30 kDa proteins. The present results suggest that the mechanisms of action of LHRH‐A and PL‐C on steroid production in Leydig cells may be similar and different from PMA, and may involve stimulation of a specific type of PK‐C or hydrolysis of a specific pool of phospholipids.

Effects of cholesterol, hydroxycholesterols and calcium in pregnenolone production rates in mitochondrial fractions from rat testes

The in vitro regulation of the mitochondrial conversion of cholesterol to pregnenolone in rat testis tissue has been further investigated. Pregnenolone production rates by isolated mitochondrial fractions could be stimulated by the addition of cholesterol. The stimulation was always highest in mitochondria isolated from lutropin-treated testes relative to control and cycloheximide treated testes. Addition 20- or 25-hydroxycholesterol resulted in a greater stimulation of pregnenolone production rates and these rates were unaffected by prior treatment with cycloheximide. When both cholesterol and 20- or 25-hydroxycholesterol were present in the incubation medium, pregnenolone production rates were mainly influenced by the hydroxycholesterol, even in the presence of a ten-fold excess of cholesterol. Ca2+ in vitro stimulated pregnenolone production rates from endogenous cholesterol as well as from added cholesterol. However, pregnenolone production rates in the presence of hydroxycholesterol were not influence by the addition of Ca2+ in vitro.

Fluorescent ligands, used in histocytochemistry, do not discriminate between estrogen receptor-positive and receptor-negative human tumor cell lines

SummaryA cell line containing estrogen receptors (MCF-7) and a cell line lacking estrogen receptors (PC-93) were used for a comparison of biochemical and histochemical procedures to detect estrogen receptors. We evaluated three different fluorescent estrogen derivatives: 17β-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, 17β-estradiol-17-hemisuccinate-fluoresceinamine, and coumestrol. The main results were: 1. The relative binding affinities of these ligands for the estrogen receptor were between 0.1 and 2% of the affinity of estradiol. 2. Fluorescent staining of the cells showed no relation to the presence of estrogen receptors. 3. Staining was not suppressed with excess estradiol- 17β, which is known to prevent binding of low affinity ligands to estrogen receptors. 4. Cells with intact membranes were not stained after treatment with the albumin-linked estrogen derivative; only cells with damaged cell membranes were stained. 5. Treatment of cells with 17β-estradiol-17-hemisuccinate-fluoresceinamine resulted in a fluorescent labeling of the cytoplasm in intact and artificially damaged cells. 6. Coumestrol caused only fluorescence of the cytoplasm in intact cells.It is concluded that estrogen receptors cannot be detected with these low affinity ligands. Fluorescence of these cells is probably due to binding of the ligands to low affinity binding sites. The presence of these low affinity binding sites appears not to be related to the presence or absence of estrogen receptors and can therefore not be used to discriminate between estrogen receptor-positive and receptor-negative tumor cells.

Further characterization of the effects of hypophysectomy, FSH and estrogen on LH stimulation of testosterone production in Leydig cells isolated from immature rats.

Hypophysectomy of immature rats results after 5 days in a loss of LH responsiveness of Leydig cells. LH responsiveness can be partly maintained by treatment with FSH for 5 days. When estradiol benzoate was administered together with FSH to hypophysectomized rats the maintenance of LH responsiveness was not observed. The loss in LH responsiveness after hypophysectomy in terms of testosterone production could not be explained by either a change in the amount of Leydig cells present in the Leydig cell preparation or to a higher conversion of testosterone. The LH-stimulated cAMP production in cells from hypophysectomized rats was very low compared to cells from intact rats. There was no difference between cAMP production of Leydig cells from untreated, FSH-treated or FSH plus estradiol benzoate treated hypophysectomized rats. During the first 2 days after hypophysectomy LH responsiveness in both untreated and FSH-treated rats showed a comparable decrease. From day 2 after hypophysectomy LH responsiveness remained at a constant level in cells from rats treated with FSH, but declined further in cells from untreated rats. A single injection of estradiol benzoate to hypophysectomized rats treated with FSH counteracted the effect of FSH on LH responsiveness, but only when estradiol was administered at that time after hypophysectomy, when the effect of FSH on LH responsiveness was clear.

Effect of cytosol fractions from lutropin-stimulated rat testes on pregnenolone production by mitochondria from normal rat testes.

The rate limiting step in the production of steroids in the testis is the mitochondrial conversion of cholesterol to pregnenolone. This conversion can be stimulated by lutropin, but the precise interaction between lutropin-induced cytoplasmic factors and the mitochondrial activity in steroid production is as yet unknown. The results described in the present paper concern the steroid production of isolated mitochondrial fractions in recombination experiments with isolated supernatant fractions from total testes homogenates. Cyanoketone as well as SU-10603, an inhibitor of steroid 17 alpha-hydroxylase activity are required to block pregnenolone metabolism. The results show that the cytoplasm contains lutropin-induced factor(s) which can exert its effect in vitro on the cholesterol side-chain cleavage activity in intact mitochondria isolated from control testes.