Effects of PRL and FSH on LH binding and number of Leydig cells in hypophysectomized mice

The effects of PRL and FSH on testicular LH receptors were studied in hypophysectomized (hypox) mice. From the eleventh day after hypophysectomy, they were given 100 micrograms ovine (o) PRL and/or 2 micrograms oFSH in two injections per day for 10 days. Hypophysectomy reduced the weight of testis, epididymides and seminal vesicles, and the LH binding to the testis. Treatment with oPRL and/or oFSH in hypox mice resulted in an increase in the weight of testis and epididymides, and the LH binding per testis. There was no difference between the testicular LH binding in oFSH- and oPRL-treated mice. Histological examination showed that oPRL and/or oFSH treatment in hypox mice restored normal spermatogenesis. Administration of oFSH to hypox mice led to an increase in the number of typical Leydig cells, whereas oPRL was not effective. These results suggest that either PRL or FSH stimulates the LH binding to the testis, but that the action of PRL and FSH on the increase in testicular LH binding is different.     

Acute N-methyl-D,L-aspartate administration stimulates the luteinizing hormone releasing hormone pulse generator in the ovine fetus

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130-142 days of gestation; term 147 +/- 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 +/- 0.36 ng/ml) and oFSH (1.21 +/- 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 microg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp6Pro9NEt-LH-RH (10 microg/day i.v. x 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D, L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10(-10) to 10(-6) M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10(-8) M; p < 0.0004) and oFSH (10(-8) M; p < 0. 0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.     

Differential effects of follicle-stimulating hormone and luteinizing hormone on Leydig cell function and restoration of spermatogenesis in hypophysectomized and photoinhibited Djungarian hamsters (Phodopus sungorus)

Effects of pure human follicle-stimulating hormone (hFSH) and ovine luteinizing hormone (oLH) on testicular function were investigated in long-term hypophysectomized or photoinhibited Djungarian hamsters. hFSH (5 IU) or oLH (5 micrograms) or a combination of FSH and LH (5 IU and 5 micrograms, respectively) were injected s.c. twice daily for 7 days to hypophysectomized and photoinhibited hamsters. Other photoinhibited hamsters were treated for 14 and 21 days with FSH and LH (3 IU and 3 micrograms, respectively) in a similar way. LH alone had little, if any, effect on testicular weights; FSH, when injected alone or in combination with LH (FSH/LH), caused a significant increase in testes weights at each time point. On the other hand, LH or FSH/LH, but not FSH alone, caused a significant increase in the accessory organ weights. FSH had no effect on intratesticular testosterone (T) or on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity but enhanced the in vitro response of interstitial cells to hCG. LH and FSH/LH had pronounced effects on intratesticular T, 3 beta-HSD activity, and in vitro response of interstitial cells to human chorionic gonadotropin. Treatment with FSH or FSH/LH caused regrowth of the testis and restoration of tubular lumen and tubular diameter and restored complete spermatogenesis. However, LH had little effect on spermatogenesis in spite of increased intratesticular and peripheral T levels. These results indicate that although LH can cause a full redifferentiation of Leydig cells in photoinhibited hamsters, it has only minor effects on tubular function. On the other hand, FSH alone induces full restoration of tubular function in these animals and has no direct effect on Leydig cell steroidogenesis, but may enhance the Leydig cell responsiveness to LH.     

Rabbit mammary prolactin receptors. Demonstration of a late puerperal increase in affinity.

Crude receptor preparations of rabbit mammary gland were made by differential centrifugation and reacted with lactoperoxidase-iodinated ovine prolactin (oPRL) in order to determine their binding characteristics. Receptors prepared from the mammary glands of animals less than 4 days postpartum bound oPRL with high affinity (Ka = 3.50 X 10(9) M-1), in good agreement with previous results of other investigators. The binding capacity of these preparations was 107 +/- 16.3 fmol/mg of protein. In contrast, receptors prepared from the mammary glands of late lactating rabbits (Days 25 to 30 of lactation) showed a 2.5-fold increase in binding affinity (Ka = 8.63 X 10(9) M-1, p less than 0.001) without a significant increase in binding capacity (135 +/- 21.4 fmol/mg, p greater than 0.2). Kinetic experiments revealed that the rates of association of hormone and receptor were identical in early and late receptor preparations, and that the 2.5-fold decrease the dissociation rate observed in the late preparations was fully explanatory of the differences in equilibrium binding. The mechanism of this affinity increase is not known. Such a change in binding characteristics, which would tend to enhance tissue responsiveness, may underlie the well characterized maintenance of full lactation in women despite falling concentrations of prolactin.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Evidence for oxytocin receptors in cultured bovine luteal cells.

Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Cultured cells from bovine corpora lutea (CL) at different stages (Days 3-5, 8-12, and 15-18 of the estrous cycle) were examined for OT receptors by a radioreceptor assay using the 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2(9)] -vasotocin. Binding specificity was demonstrated in displacement studies with various related peptides. Scatchard analysis revealed the presence of a binding site with an association constant of Ka = 2.6 x 10(9) M-1 and a capacity of 5.9 fmol/micrograms DNA. Additionally, in 50% of the experiments (n = 6) two different binding sites were observed. The Ka of the high-affinity site was 2.6 x 10(10) M-1; its capacity was 0.73 fmol/micrograms DNA. The low-affinity site had an apparent Ka of 4.9 x 10(8) M-1 and a capacity of 8.8 fmol/micrograms DNA. Observation of one versus two binding sites related neither to the assay conditions nor to the state of the individual CL used for the cell culture and therefore appeared to reflect individual variation within the OT receptor population. Significant binding of OT was observed at all luteal stages. OT binding was maximal at the mid-luteal stage (Days 8-12). We conclude that a direct action of OT on the bovine CL is mediated by the OT receptor, supporting the hypothesis that luteal OT plays an important physiological role in the regulation of progesterone release and/or other luteal functions in a paracrine or autocrine fashion.     

Properties of equine luteinizing hormone alpha subunit alone and in combination with various beta subunits

Previous studies have shown that equine luteinizing hormone (eLH) inhibits production of cyclic adenosine monophosphate (cAMP) induced by follicle-stimulating hormone (FSH) in preparations of seminiferous tubules from immature rats. It was also shown that the inhibitory effect was a function of the equine LH (eLH) alpha subunit. To explore this phenomenon further, the intrinsic FSH-like activities of eLH alpha alone and in combination with ovine (o) LH beta, ovine FSH beta, and equine FSH beta were evaluated in several assay systems. In a radioreceptor assay employing 125I-o-FSH and testis membranes from day-old calves, eLH was twice as active as oFSH, eLH alpha was 6% as active as oFSH, and other subunits showed a lack of activity (less than 1.5%). Whereas oLH was only 0.1% as active as oFSH, the hybrid eLH alpha-oLH beta was 3.0% as active. The binding activity of eLH alpha-FSH beta hybrids tended to be higher than the oFSH alpha-FSH beta hybrids. In the cAMP production assay, eLH alpha-FSH beta hybrids exhibited dampened dose-response curves when compared to the oFSH alpha-FSH beta hybrids. In a plasminogen activator assay (PAA) employing granulosa cells from intact 21-24-day-old female rats primed with diethylstilbestrol, eLH had activity comparable to that of oFSH, while eLH alpha was inactive. When eLH alpha was recombined with oFSH beta, eFSH beta, or oLH beta, the PAA stimulatory activity was not altered compared to that of the hybrids oLH alpha-oFSH beta, oFSH alpha-eFSH beta, and the recombinant oLH alpha-oLH beta, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of testicular and Sertoli cell gamma-glutamyl transpeptidase by follicle-stimulating hormone

Hormonal deprivation achieved by hypophysectomy or gonadotropin-releasing hormone (GnRH)-antagonist treatment of immature rats resulted in markedly lower testicular gamma-glutamyl transpeptidase (GGT) activity than in the testes of age-matched controls. When begun 15 days after hypophysectomy, follicle-stimulating hormone (FSH) treatment significantly increased testicular GGT above that in testes from hypophysectomized controls in a time- and dose-dependent manner. In contrast, testosterone propionate had only a small effect. Testicular GGT was higher in adult hypophysectomized rats treated with FSH from the time of surgery than in untreated hypophysectomized rats; testosterone propionate treatment had no effect. GGT activity in Sertoli cells isolated from GnRH antagonist-treated or hypophysectomized immature rats was also lower than in cells from control rats. FSH treatment from the day of hypophysectomy resulted in Sertoli cell GGT values equivalent to those from intact controls. These data indicate that FSH regulates GGT activity in rat testis and Sertoli cells.     

Endocytosis of follicle-stimulating hormone by ovarian granulosa cells: analysis of hormone processing and receptor dynamics

Suspensions of freshly isolated rat granulosa cells were used to study endocytosis and processing of radioiodinated ovine follicle-stimulating hormone (I-oFSH) and to analyze the dynamics of its receptor. Ovine FSH was iodinated to a specific activity of 26 microCi/micrograms as determined by radioreceptor self-displacement assays with maximum specific binding to excess membrane receptors of 46%. Radiolabeled oFSH was judged biologically equivalent to the unlabeled hormone since I-oFSH shows saturation-binding kinetics and stimulates steroidogenesis in a similar dose-related manner to unlabeled oFSH. Experiments designed to study the extent and time course of degradation involved continuous exposure of isolated granulosa cells to I-oFSH. Saturation of membrane receptors was achieved within 1.5 h of incubation, and internalization of FSH occurred in a linear manner for up to 6 h. The rate of internalization was equivalent to 2,780 FSH molecules/cell/h. Degradation of FSH became apparent after 6 h of incubation and increased to 86% of total cellular-associated radioactivity at 22 h. FSH degradation was inhibited by 100 microM chloroquine or 0.45 mM leupeptin. The measurement of cell surface I-oFSH binding in the combined presence of 100 microM chloroquine and 0.5 mM cycloheximide was unchanged for up to 22 h of incubation. This and other receptor binding data suggest that there is no reutilization of FSH receptors. Scatchard analyses of 4 degrees C binding assays on intact cells indicated that a two-site model best fit the data with association constants of K11 = 1.44 (+/- .42) X 10(10) and K12 = 4.35 (+/- .91) X 10(8). Receptor binding and activation studies for progesterone production yielded ED50s of 270 pM and 7.7 pM, respectively, and also indicated that 20% receptor occupancy is sufficient to stimulate maximal progesterone production. We conclude that after the initial binding event, FSH is endocytosed very slowly and is subsequently shuttled to the lysosomal compartment for degradation. The retarded rate of endocytosis may relate to novel pathways of hormone processing.     

Binding of triiodothyronine to the nuclear matrix of the rat liver. The effect of thyroid hormones on the phosphorylation of nuclear matrix proteins

The interaction of thyroid hormones with rat liver nuclear matrix proteins was studied. It was shown that the nuclear matrix contains the sites which bind triiodothyronine with a high affinity (Ka = 1.07 X 10(9) M-1) and limited capacity (maximal binding capacity--28.5 fmol triiodothyronine/100 micrograms protein). Electrophoretic analysis of triiodothyronine-binding matrix proteins revealed that the molecular mass of the major triiodothyronine-binding fraction is 50 000-52 000 Da. Injections of triiodothyronine to thyroidectomized animals stimulated the phosphorylation of all protein fractions of the nuclear matrix.     

Charge microheterogeneity of ovine follicle-stimulating hormone in rams and steroid-treated wethers

Chromatofocusing was utilized to separate the isohormones of ovine follicle-stimulating hormone (oFSH) in pituitary extracts from rams, wethers, and wethers receiving Silastic implants of dihydrotestosterone (DHT), 17 beta-estradiol (E2), or both DHT and E2. Pituitary extracts were prepared by homogenization and centrifugation at 100,000 x g. Extracts of ram pituitaries yielded at least nine species (isohormones) of immunoreactive oFSH having apparent isoelectric points (pIs) of greater than 7.40, 6.74, 6.52, 5.76, 5.20, 4.74, 4.44, 4.10, and less than 4.00. These isohormones were designated by the letters A-H and Z, respectively. Eighty-four percent of the immunoreactive ram oFSH recovered from the chromatofocusing column was very acidic in nature, having apparent pIs less than 5.30. The majority of the immunoreactive oFSH was focused in an area different from that of most of the immunoreactive ovine luteinizing hormone (oLH) and ovine thyroid-stimulating hormone (oTSH). Pituitary extracts from control and steroid-treated wethers also contained these nine oFSH isohormones, but significant differences were noted in the relative distribution among the isohormones. Castration resulted in a 4-fold increase in isohormone B and a concomitant reduction in isohormones E and H. DHT administration returned these levels to the values observed in the ram, whereas E2 administration produced a significant 2-fold increase in the most acidic form (isohormone Z). The implant combination produced isohormone profiles comparable to that of E2 alone. Neuraminidase treatment in vitro of both crude pituitary extracts or highly purified iodinated oFSH abolished the most acidic form and caused a marked shift in the isohormone pattern to more basic species. These results demonstrate that 1) at least nine isohormones of oFSH can be separated reproducibly from the male ovine pituitary by chromatofocusing, 2) the majority of FSH in the pituitary exists in acidic form, 3) castration and steroid administration alter the distribution of oFSH in the pituitary among its isohormones, and 4) at least a portion of oFSH-charge microheterogeneity appears to be due to the presence of sialic acid residues on the molecule.     

FSH-induced ovulation in intact and hypophysectomized mice

A single, ovulatory dose of 25 micrograms of a highly purified preparation of ovine FSH caused ovulation in 89% of hypophysectomized and 91% of intact female mice primed 48 h earlier with PMSG; the number of oocytes recovered (29.4 +/- 4.7 and 22 +/- 2.7/mouse ovulating, respectively) compared favourably with the 20.0 +/- 2.9 oocytes per ovulating female recovered from animals that received PMSG + hCG. After oFSH injection, 82% of oocytes released were fertilized and developed to blastocysts. That the trace contamination (less than 0.2%) of the oFSH with oLH was not responsible for the ovulation was shown by the markedly reduced number of oocytes collected from ovulating females that were injected with equivalent low levels of hCG (0.001 micrograms) or oLH (1 microgram) (9.0 +/- 3.3 and 8.0 +/- 3.1, respectively). These results demonstrate that oFSH is as effective as LH in inducing ovulation of competent oocytes in the mouse.     

Basic properties and annual changes of follicle-stimulating hormone receptors in the testis of horseshoe bats, Rhinolophus ferrumequinum

The unique reproductive patterns, delayed fertilization in females, and asynchrony between spermatogenesis and mating behavior in males are well documented in bats living in temperate latitudes. The present study was undertaken to examine follicle-stimulating hormone (FSH) receptors in the testis of bats, Rhinolophus ferrumequinum, during the annual reproductive cycle. Male bats were captured at natural roosting sites and testicular preparations were subjected to a radioligand binding assay for FSH receptors. The weight of paired testes increased considerably in the spermatogenic period and decreased from the mating to hibernation periods. Meiotic division in the testis was observed in the spermatogenic period but not the mating period. Serum testosterone concentrations increased in the spermatogenic period and rapidly decreased in the mating period. The binding of FSH was specific for mammalian FSHs and detected primarily in the testis. Scatchard plot analyses of the binding of FSH to bat testicular preparations showed straight lines, suggesting the presence of a single class of binding sites. The affinities (equilibrium association constant) of FSH receptors were consistent throughout the annual reproductive cycle. The specific binding per unit weight of testis and total binding in the paired testes were highest in the mating period and in the spermatogenic period, respectively, among reproductive periods. The accumulation of cyclic adenosine 3', 5'-monophosphate to FSH stimulation was higher in the spermatogenic period than in the hibernation period. These findings suggest that testicular function of bats is associated with seasonal changes in the number of binding sites, while the number per target cell and the activation of adenylate cyclase led by FSH-receptor complex considerably decreases in the hibernation period.     

Prolactin receptors in the hypoprolactinemic male rat (IPL nude rat): measurement in testis and induction in liver

In order to evaluate the importance of PRL in the regulation of its own receptors, characteristics of specific binding for PRL were studied in membrane preparations from liver and testis of a new hypoprolactinemic male rat, the IPL nude male rat, and this was compared to those found for normal male rats. Under basal conditions, hepatic specific binding of PRL in IPL nude rats, as in normal rats was not detectable. Following castration, it became detectable in both groups, and was 6.99 +/- 0.78% and 6.34 +/- 0.87% for IPL nude and normal rats respectively. Under such conditions, the apparent affinity constant (Ka) and the binding capacity (Nmax) obtained were also similar for both groups (Ka) = 1.36 +/- 0.14.10(9) M-1, Nmax = 102 +/- 14 fmol/mg protein in IPL and Ka = 1.34 +/- 0.28.10(9) M-1, Nmax = 97 +/- 11 fmol/mg protein in normal rats) although a decrease in serum levels of PRL was observed in both groups. This decrease was greater for IPL nude rats. As already reported, estradiol injection following castration was able to further increase the percentage of PRL hepatic specific binding (4 times). Furthermore, our results demonstrated that the affinity constant was significantly increased by estradiol injection in both groups. On the other hand, for testicular PRL binding characteristics, a statistically significant difference was found between IPL nude and normal rats. The PRL specific binding percentage was 7.01 +/- 0.85% for the IPL nude rat and 10.07 +/- 0.64% for the normal rat. By Scatchard analysis, the Ka of testicular membranes for labelled oPRL was similar in both groups, while the capacity differed (Nmax = 9.82 +/- 1.25 fmol/mg protein for IPL nude rat and Nmax = 26.06 +/- 4.39 fmol/mg protein for normal rats). These data established the fact that IPL nude male rats presented characteristics of hepatic PRL receptors similar to those of normal rats, while their testicular oPRL binding significantly differed. These findings therefore suggest that in genetic hypoprolactinemic rats (IPL nude rats), PRL might be more involved in the regulation of testicular PRL receptors than in that of hepatic receptors.     

Properties of the binding of follicle-stimulating hormone to the fetal rat testis

Basic properties of the binding of [131I]-labeled rat FSH ([131I]rFSH) to the testicular homogenates of fetal rats were analyzed by micro-radioreceptor assay. Specific binding of FSH was detectable in the testicular preparations from 15.5-day fetuses, but it was very low. After 17.5 days of gestation, specific FSH binding was apparent in the testis and was effectively displaced by rat FSH but not by rat LH. The Scatchard plot analyses of the binding of FSH to the testicular preparations of fetuses showed straight lines similar to those of postnatal rats, suggesting the presence of a single class of binding sites. The mean dissociation constant (Kd) for FSH receptors in 17.5-day fetuses was 0.413 +/- 0.043 nM, which was significantly greater than that in postnatal rats at 50 days of age. However, the Kd in 19.5-day fetuses was not significantly different from those in 17.5-day fetuses and postnatal rats due to its considerable variance. The capacity of FSH binding sites was 0.51 +/- 0.01 fmol/testis in 17.5-day fetuses, which was significantly less than those of 19.5-day fetuses and postnatal rats.     

Lutropin receptors from male and female tissues: different responses to a lutropin receptor binding inhibitor.

An inhibitor for lutropin receptor site binding (LH-RBI), which strongly inhibited the binding of 125I-labeled ovine lutropin ([125I]oLH) to ovarian LH receptors, did not inhibit the [125I]oLH binding to testicular LH receptors. Preincubation of the LH-RBI with [125I]oLH did not affect the binding of preincubated ]125I]oLH to ovarian LH receptors. No inhibition of [125I]oLH binding to testicular LH receptors was observed even uhen the concentration of LH-RBI was significantly increased or when the testicular LH receptors uere first incubated with LH-RBI prior to the addition of [125I]oLH and a second incubation. Scatchard analysis revealed that the dissociation constant of [125I]oLH binding was essentially the same in the presence or absence of LH-RBI. The results suggest that: (i) the lutropin receptor of ovaries, but not of testes, has a specific LH-RBI binding site in addition to the lutropin binding site, and (ii) the binding of the LH-RBI produces an "allosteric" type of inhibition to the binding of lutropin at the lutropin binding site.     

Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes

An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.     

Sheep testicular gonadotropin binding sites: characterization and changes with surgical shortening of the scrotum

Gonadotropin receptors in previously frozen (-70 degrees C) sheep testicular tissue were characterized, and methods of assessment of receptor binding activity were established and applied to an investigation of testicular function in the short scrotum ram. Binding of 125I-labelled ovine luteinizing hormone (125I-oLH) and 125I-labelled ovine follicle-stimulating hormone (125I-oFSH) to testicular membranes was highly specific and saturable. Uptake of labelled gonadotropins was proportional to the amount of membrane protein, with 125I-oFSH showing greater specific binding. Initial association of 125I-oLH with binding sites was comparable at 4, 25, and 34 degrees C; with prolonged incubation, maximal binding occurred at 4 degrees C. Equilibrium was achieved in 8 h at 34 degrees C and in 16 h at 25 and 4 degrees C. In contrast, the temperature-dependent association of LH with rat testicular membranes was greater at 25 than at 4 degrees C. The rate of association of 125I-oFSH to binding sites was proportional to incubation temperature, with equilibrium being achieved in 2 h at 34 degrees C and in 16 h at 25 degrees C; binding at 4 degrees C; was slow and still increasing by 48 h. Binding of radioactive and nonradioactive oLH and oFSH was hormone specific and increased in a dose-dependent manner until saturation occurred. Shortening the scrotum of adult rams led to reductions (p less than 0.05) in testicular weight (60%) and in the number of LH (55%) and FSH (90%) binding sites per testis, with no apparent change in serum testosterone concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro binding of radioiodinated rat follicle-stimulating hormone to the testis of the mallard duck, Anas platyrhynchos l

The binding of 125I-labelled rat FSH to homogenates and frozen sections of mallard duck testis was investigated. The equilibrium association constant (Ka) in the homogenates (8.5 x 10(9) M-1) was similar to those reported in other avian and mammalian species. Autoradiography suggested that the binding sites for the labelled hormone were localized in the tubular compartment.     

Effect of cyanoketone on follicle-stimulating hormone (FSH) induction of receptors for FSH in granulosa cells of the rat

Follicle-stimulating hormone (FSH) enhances the conversion of testosterone or androstenedione into estradiol by stimulating the aromatase enzyme system. Estradiol also enhances FSH action. Thus, a synergistic action of FSH and estradiol may be required for maturation of ovarian follicles. We hypothesized that estradiol may be required for FSH action. Thus, blocking estrogen synthesis should prevent FSH-induced increases in FSH receptors. Hypophysectomized rats were divided into five groups and injected subcutaneously with: 1) saline, 2) cyanoketone (0.05 mg, blocks the conversion of pregnenolone to progesterone), 3) ovine FSH (oFSH, 200 micrograms), 4) cyanoketone then oFSH 24 h later, or 5) cyanoketone plus estradiol [or progesterone, testosterone, promegestrone (R5020), dihydrotestosterone (DHT), 2 mg], then FSH 24 h later. Animals were decapitated at 0, 12 or 24 h after an injection of oFSH, and membrane receptors for FSH and luteinizing hormone (LH), plus nuclear receptors for estradiol from granulosa cells, were measured. LH receptor levels were increased only after administration of FSH and estradiol. At 0 and 24 h, numbers of FSH or estradiol receptors were similar in saline- and cyanoketone-treated animals. FSH alone increased (P less than 0.01) FSH and estradiol receptors 3-fold and 4-fold, respectively, over controls by 12 and 24 h. Cyanoketone prevented these increases in FSH and estradiol receptors. Estradiol replacement fully reversed the effects of cyanoketone on FSH action. Replacement with progesterone and testosterone was able to only partially restore levels of FSH receptors; however, estradiol receptor numbers were also increased.(ABSTRACT TRUNCATED AT 250 WORDS)