Properties of follicle-stimulating-hormone receptor in cell membranes of bovine testis.

A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 X10(-11)M, and 5.9 X 10(-14)mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40 degrees C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5mu/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone.     

Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

Summary In testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7–10 and increased thereafter. The uptake of ¹²⁵I-labelled human follicle-stimulating hormone (¹²⁵I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of ¹²⁵I-labelled human choriogonadotropin (¹²⁵I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7–10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.     

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.     

Binding of 125I-labelled FSH in the mouse testis in vitro.

In-vivo testicular binding of highly purified pituitary FSH, labelled by a method which did not significantly affect biological potency, was hormone-specific, tissue-specific and dose-dependent. Hypophysectomy of mice was followed by a progressive increase in the amount of 125I-labelled FSH per unit weight of testis but not in the total amount of hormone taken up by the testis. Maximum binding occurred at 4 h in a membrane-containing fraction prepared by high-speed ultracentrifugation of testicular homogenate. This is considerably later than has been reported for testicular tissue incubated in vitro at 37 degrees C.     

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)     

Testicular FSH receptor numbers and affinity in bulls of various ages

Bulls (N = 42) ranging in age from 1 day to 5.5 years were used to determine whether a change in the concentration of FSH receptors in the bovine testis occurred as bulls matured. 125I-labelled human FSH was used as the ligand to evaluate binding to bovine testicular membranes. Membrane fractions were collected by centrifugation of testicular homogenates at 120 g and recentrifugation of the 120 g supernatant at 1250 g. Relative binding activity of membrane sedimented at 1250 g was determined after incubation of membranes with 125I-labelled FSH for 16--18h at 25 degrees C, followed by centrifugation (1250 g) to separate bound from free hormone. Specifically bound FSH when expressed as fmol/mg protein was negatively correlated with age (r = -0.73). The association constant (Ka) determined by Scatchard analysis was the same for bulls at all ages with a mean (+/- s.e.m.) Ka = 1.5 +/- 0.3 x 10(9) M-1. Concentration of FSH receptors on a per mg protein basis declined rapidly from birth to 2.5 years of age and remained low up to 5.5 years of age. On a whole testis basis the total number of receptors increased as the bulls matured. After 2.5 years of age total testicular binding did not change.     

Identification of a receptor binding region on the beta subunit of human follicle-stimulating hormone

Mouse epidermal growth factor (mEGF) and the beta subunit of follicle-stimulating hormone (hFSH) (hFSH-beta) have been shown to inhibit binding of intact hFSH to its testes membrane receptor in vitro. Both hFSH-beta and mEGF contain the tetrapeptide sequence Thr-Arg-Asp-Leu (TRDL). Previous results demonstrated that synthetic TRDL inhibited binding of intact hFSH to receptor. We therefore investigated the possibility that TRDL was located on an exposed region of FSH-beta using a polyclonal antiserum to hFSH [NHPP anti-hFSH batch 4 (AB4)] which recognized determinants on intact hFSH and its beta subunit, but not the alpha subunit. Pituitary FSH preparations from several mammalian species produced parallel inhibition curves in a heterologous [AB4 and 125I-labeled ovine FSH (125I-oFSH)] radioimmunoassay with relative potencies similar to those observed for the same preparations assayed by radioligand receptor assay. This antiserum also competitively inhibited 125I-FSH binding to receptor. Thus, AB4 appeared to recognize antigenic determinants that are highly conserved and located at or near regions involved with hormone recognition of receptor for FSH. Synthetic TRDL inhibited 50% of 125I-hFSH binding to antiserum at a concentration of 1.36 mg/tube (9 x 10(-3) M). Other tetrapeptides (Thr-Pro-Arg-Lys and Lys-Thr-Cys-Thr) had no inhibitory activity at comparable concentrations. A mixture of the free amino acids T, R, D, and L inhibited radioligand binding only at significantly higher concentrations than TRDL.(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.     

Impaired gonadotrophin uptake in vivo by the cryptorchid rat testis

The specific testicular uptake in vivo of 125I-labelled hCG was compared in control adult rats and adult rats made bilaterally cryptorchid 5 weeks previously. Although a similar temporal pattern of uptake was observed in both groups, uptake of hCG by cryptorchid testes was reduced at all times after injection by up to 70%. The possible causes of this impairment were investigated. It could not be accounted for by differences in the rate of absorption or clearance of 125I-labelled hCG in the two groups. Therefore, because hCG-induced increase in the permeability of testicular capillaries is a crucial factor in determining hCG uptake by the testis, this change was compared in control and cryptorchid testes. Although hCG induced a characteristic increase in testicular capillary wall permeability in both groups, this change was temporally delayed in cryptorchid testes, and occurred after hCG values in the blood had fallen. Even when hCG had crossed the capillary wall into testicular interstitial fluid, its uptake into the testicular tissue was significantly lower in cryptorchid than in control testes. These changes probably account for the impairment of gonadotrophin uptake by the cryptorchid testis and have important implications with respect to the aetiology of Leydig cell changes in cryptorchidism.     

Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate

At 37 degrees C 125I-labelled human (h) FSH (NIAMDD-hFSH-I-3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Binding of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1-2.5 mm vs greater than or equal to 3 mm). Irrespective of breed, genotype or follicle size, the mean (+/- s.e.m.) calculated association rate constant, (ka) was 7.3 (+/- 0.8) x 10(5) litres mol-1 sec-1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was less than 5% after 30 min and low but variable (i.e. between 0 and 30%) after 2-6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2.5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (less than or equal to 0.1%). The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1-2.5 mm; or greater than or equal to 3 mm). The overall mean +/- s.e.m. (n = 24) Bmax and Kd values were 16.7 +/- 0.8 fm/mg protein (i.e. approximately 800 available receptor binding sites/cell) and 1.1 +/- 0.1 nM respectively. Collectively, these findings suggest that the earlier maturation of follicles in FF or F+ ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.     

Temporal changes in rat Leydig cell function after the induction of bilateral cryptorchidism

Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.     

Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein

We have previously reported detergent (Triton X-100) solubilization of a follitropin (FSH) receptor-rich fraction from light membranes of bovine testis that responded to exogenous FSH by activation of adenylate cyclase (Dattatreyamurty, B., Schneyer, A., and Reichert, L. E., Jr. (1986) J. Biol. Chem. 261, 13104-13113). Upon gel filtration of the detergent-extract through Sepharose-6B, two fractions were separated. Each specifically bound [3H]guanosine 5'-imidotriphosphate (Gpp(NH)p) and had guaninetriphosphatase (GTPase) activity. Of these, one fraction (6B-Fraction-1) also bound radioiodinated human follitropin (hFSH), indicating a coelution of the nucleotide-binding protein with receptor. The other fraction (6B-Fraction-2) did not contain detectable FSH receptor activity. Several lines of evidence suggest that 6B-Fraction-1 is a complex consisting of FSH receptor and a guanine nucleotide regulatory protein, probably Ns. 1) The GTP-binding and FSH-binding activities of 6B-Fraction-1 were retained by a GTP-affinity column, and their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p. 2) When exogenous GTP was added to 6B-Fraction-1, binding of 125I-hFSH was reduced compared to controls lacking exogenous GTP. This effect of GTP was highly specific and noncompetitive, indicating that GTP did not bind to receptor. In addition, the affinity of receptor for FSH was decreased, and the rate and degree of dissociation of bound labeled FSH from receptor were increased in the presence of exogenous GTP, each in concentration-dependent manner. 3) Exposure of 6B-Fraction-1 to higher concentration of Triton X-100 reduced significantly the receptor-associated GTP-binding activity and also rendered the hormone-binding activity insensitive to GTP. 4) Treatment of highly purified testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminated the ability of GTP to modulate the 125I-hFSH binding to receptor. 5) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a major peak of radioactivity (presumably Ns) was coeluted with FSH receptor activity from the Sepharose-6B column. These results and the observation that the effect of GTP is noncompetitive at FSH receptor level suggest that FSH binding inhibition and the increased rate of hormone dissociation from receptor were the result of GTP interaction with a guanine nucleotide regulatory protein, probably Ns, which itself was functionally associated with the FSH receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of different batches of 125iodine on properties of 125I-hFSH and characteristics of radioligand-receptor assays

Radioiodination of highly purified human follicle-stimulating hormone (hFSH) (4000 IU/mg) was performed every other week for 23 weeks using 2 mCI carrier free Na125I (Amersham Corp., 15 mCi/micrograms I2) in the presence of lactoperoxidase. Incorporation of 125I into hFSH was determined by the method of R. C. Greenwood, W. M. Hunter, and J. S. Grover (1963) Biochem. J. 89, 114). Hormone binding was studied in vitro under steady-state conditions (16 h, 20 degrees C) using different calf testis membrane preparations having similar receptor characteristics. Each 125I-hFSH preparation was characterized for maximum bindability, specific activity of bindable radioligand as determined by self-displacement analysis, and by determination of Ka and Rt. Incorporation of 125I into FSH was relatively constant over the large number of experiments (62.4 +/- 6.4 microCi/micrograms; n = 23). By comparison, however, specific radioactivity of the receptor bindable fraction of 125I-hFSH was related to the lot of 125I utilized, and was significantly (P less than or equal to 0.01) lower and more variable (28.7 +/- 10.5 microCi/micrograms). Maximum bindability of 125I-hFSH was not correlated to specific activity (r = 0.06) but was negatively correlated to hFSH 125I incorporation (r = -0.47; P less than or equal to 0.05). These observations demonstrate the need to assess the quality of each batch of radioligand before undertaking radioligand-receptor assays and suggest that differences in Na125I lots affect specific radioactivity of the radioligand and its receptor binding characteristics.     

Attempt to map the receptor binding sites of human follicle-stimulating hormone using disulfide peptides of its beta-subunit indicates major involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone.

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone secreted by the anterior pituitary. It plays a very important role in folliculogenesis in females and is responsible for spermatogenesis in males. The alpha-subunit which is common within a species and the beta-subunit which is hormone-specific are held together by noncovalent association. This association is very essential for the biological activity of the hormone. Each of these subunits are highly cross-linked by disulfide bonds which appear to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This study was initiated to delineate the role of the disulfide bonds of hFSH beta in receptor binding of the hormone. Five intermolecular and one intramolecular disulfide peptides corresponding to the disulfide bonds found in hFSH beta were synthesized and screened along with their linear counterparts, for their ability to competitively inhibit the radiolabelled [125I]hFSH from binding to the FSH receptor containing membranes from the testis of immature rats. The disulfide peptides Cys28-Cys82 and Cys32-Cys84 were found to be the most potent in inhibiting radiolabelled hFSH from binding to its receptor. The results suggest the involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone. The studies also suggest the involvement of beta L2 and beta L3 loop regions in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hFSH.     

Human chorionic gonadotropin binding to rat testis receptors is inhibited by a thymus factor

An interrelationship between immune and reproductive systems has been postulated, and involves, among others, bidirectional effects between gonads and thymus. To this effect a rat thymus fraction of about 28000 mol wt has been reported to inhibit the effect of hCG on in vitro suspension of Leydig cells. We have investigated the antigonadotropin activity of thymus extracts on rat testis receptors. Acetonic powder obtained from thymus of 14 day-old rats was separated by molecular sieve chromatography. The effect of the collected fractions on the 125I-hCG binding to receptor sites in rat testes was evaluated. A fraction corresponding to 27000-28000 mol wt named thymus factor (TF), was found to inhibit the binding activity of 125I-hCG to its testicular receptor. The inhibitory effect of TF on hCG binding is dose related. By Scatchard analysis a competitive interaction at the receptor level between TF and hCG was demonstrated. The Ka values of hCG binding were diminished in the presence of TF while no significative changes were detected in the number of receptor sites. Present results strongly suggest a modulation function of TF at the testis receptor level.     

The subunit structure of the follitropin receptor. Chemical cross-linking of the solubilized follitropin-receptor complex

Homobifunctional cross-linkers were utilized to characterize high affinity (Ka = 2.2 X 10(-10) M-1) follitropin (FSH) receptors in immature bovine testis. Following the formation of radioiodinated human FSH (125I-hFSH)-receptor complexes, the membranes were solubilized with Triton X-100 or beta-octyl glucoside and the supernatants from ultracentrifugation (220,000 X g) subjected to gel filtration (Sephadex G-200) to separate the labeled hormone-receptor complexes from the unbound 125I-hFSH. The appearance of a high molecular weight (greater than or equal to 200,000) radioactive component in the elution profile was abolished when an excess of unlabeled hFSH was included in the initial incubation. After concentration by ultrafiltration, the 125I-hFSH-receptor complex, as well as the free hormone, was treated with a variety of chemical cross-linkers and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Bands of Mr = 65,000 and 83,000 observed in the autoradiograph of the hormone-receptor complex was not present in autoradiographs of free 125I-hFSH, nor were they present when an excess of unlabeled hFSH was included in the initial binding incubation mixtures. The 65,000 and 83,000 Mr bands were, therefore, considered to represent cross-linked complexes of labeled hFSH (Mr = 38,000) or its subunits (hFSH alpha, Mr = 16,000; hFSH beta, Mr = 21,000) and components of the FSH receptor. The bands were observed on autoradiographs when the extraction of the membranes was performed with either Triton X-100 or beta-octyl glucoside and when cross-linking was accomplished with disuccinimidyl suberate, ethylene glycol bis(succinimidyl succinate), or bis[2-(succinimido oxycarbonyl)oxyethyl]sulfone. The Mr of the native FSH receptor in the calf testis has been estimated at 146,000. Our studies demonstrate the multimeric nature of the FSH receptor. However, FSH is also composed of subunits, so that due to the complexity of the system, it was not possible to arrive at a precise assessment of the Mr or quaternary structure of the receptor subunits.     

Determination of substances inhibiting and stimulating the binding of FSH to its receptors

We describe a method to estimate binding of human 125I-FSH to a preparation of bovine testicular receptors. Various experimental conditions are tested and the validity of the method is demonstrated. Using this method, the presence of biological substances modifying the FSH binding is measured in various fractions of ram retetestis fluid submitted to several steps of purification by chromatography. FSH receptor binding inhibitor (FSHRBI) activity is obtained in a low molecular weight fraction and FSH receptor binding stimulator activity in a larger one. These cybernin activities are isolated in fractions different from the ones observed with inhibin and GnRH like activities.     

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.     

Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1‐positive subpopulations in men

Background Isolation of spermatogonial stem cells (SSCs) could enable in vitro approaches for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. SSC isolation from adult testes is difficult due to low cell numbers and lacking cell surface markers. Glial cell‐derived neurotrophic factor family receptor alpha‐1 (GFRα1) plays a crucial role for the maintenance of SSCs in rodents and is expressed in monkey spermatogonia. Methods Magnetic activated cell sorting was employed for the enrichment of GFRα1+ spermatogonia from adult primate testes. Results Magnetic activated cell sorting of monkey cells enriched GFRα1+ cells threefold. 11.4% of GFRα1+ cells were recovered. 42.9% of GFRα1+ cells were recovered in sorted fractions of human testicular cells, representing a fivefold enrichment. Interestingly, a high degree of morphological heterogeneity among the GFRα1+ cells from human testes was observed. Conclusions Magnetic activated cell sorting using anti‐GFRα1 antibodies provides an enrichment strategy for spermatogonia from monkey and human testes.