Effect of transforming growth factor-beta on human chorionic gonadotropin induced testosterone production by cultured rat testicular cells

The potential role of transforming growth factor-beta (TGF-beta) as an intragonadal regulator in the testis was investigated by studying the effect of TGF-beta on testosterone (T) production by neonatal rat testis cells in primary cultures. After 3 days of preincubation in serum-free medium, testis cells were treated with hormones for 3 additional days. Human chorionic gonadotropin (hCG) treatment (0.3-30 ng/ml) of testis cells elicited a dose-dependent increase of T levels with maximum values greater than 9-fold over baseline. Although TGF-beta alone did not affect T levels, a dose-dependent inhibition of hCG-stimulated T production was observed when cells were cotreated with TGF-beta. Maximal inhibition was greater than 85%, and the IC50 value was 5 ng/ml (2 x 10(-10) M; n = 5 experiments). This inhibitory effect was evident 48 h after the initiation of treatment and could be reversed 1 day after the cessation of TGF-beta exposure of cells. TGF-beta also reduced forskolin and (Bu)2cAMP-induced T production (greater than 85% decrease), indicating that TGF-beta can inhibit steroidogenesis distal to the formation of cAMP. The conversion of exogenously added androgen precursors (progesterone (P) and 17 alpha-hydroxyprogesterone) to T by hCG-stimulated cells was suppressed by the addition of TGF-beta. In contrast, endogenous P accumulation did not change in cultures treated with TGF-beta. Because TGF-beta-like activity has been found in the testis, the observed inhibitory effect of TGF-beta suggests a potential intratesticular regulatory role of this growth factor.     

Slow internalization of human chorionic gonadotropin by cultured granulosa cells

Kinetic studies were performed on two-day cultures of rat ovarian granulosa cells to follow the fate of surface-bound 125I-labeled human chorionic gonadotropin (125I-hCG). Low pH was used to release hCG from its surface receptor, allowing us to distinguish between surface-bound and internalized hormone. Because our results indicated that hormone is lost from the cell surface by dissociation as well as internalization, equations were derived to determine independent rate constants for each process. We calculate that if hormone binding were irreversible, the t 1/2 for internalization would be 8.5 hour. Morphometric studies on the uptake of horseradish peroxidase indicate that the t 1/2 for internalization of bulk membrane in granulosa cells is 55 to 77 minutes. Thus, the rate of uptake of surface-bound hCG appears to be seven to nine times slower than the rate of uptake of bulk plasma membrane, which suggests that the LH/hCG receptor may be selectively excluded from the endocytic vesicles of granulosa cells.     

Regulation of serum testosterone in men with steroid sulfatase deficiency: response to human chorionic gonadotropin

Human chorionic gonadotropin (hCG; 5000 IU) was administered to 6 control men and 6 patients with recessive x-linked ichthyosis (RXLI) with verified 3 beta-hydroxysteroid sulfate sulfatase (3 beta-HSS) deficiency in their skin biopsy samples. Concentrations of steroids and their sulfate conjugates were determined in peripheral serum specimens collected a day before and 4 days after hCG administration. Testosterone concentrations were identical in patients and controls. Baseline serum LH concentrations were also identical in the 2 groups showing that there were no major differences in the regulation of the hypothalamic-pituitary-gonadal axis. The significantly increased (31-82%) serum concentrations of sulfated pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and 5-androstene-3 beta,17 beta-diol in patients compared with controls indicated that their circulating concentrations were regulated by 3 beta-HSS. This is in line with the fact that the baseline concentrations of the same unconjugated steroids were significantly lower (32-90%) in patients with RXLI, suggesting that a proportion of these circulating steroids were derived from the corresponding sulfated precursors. The response patterns and actual concentrations of testosterone, 17-hydroxyprogesterone and estradiol were similar in the patients and the controls after hCG. The decreased concentrations of testosterone sulfated at carbon 17 under baseline conditions and after hCG in patients with RXLI remains enigmatic. In conclusion, testosterone production and the response to hCG seem to be identical in patients with RXLI and controls despite the fact that significant differences were observed in the circulating concentrations of several unconjugated and sulfated testosterone precursors.     

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells. Neither growth hormone which was reported previously to stimulate hepatic 5 alpha-reductase activity, nor insulin, insulin-like growth factor-I, or epidermal growth factor, which have been reported to modulate Leydig cell function, had any effect on 5 alpha-reductase activity of Band 3 cells. These studies suggest that the major factor directly stimulating 5 alpha-reductase activity in Leydig cells during early maturation is LH. However, it is possible that other factors acting indirectly may modulate the maturational rise in 5 alpha-reductase activity.     

Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Human leukocyte interferon inhibits human chorionic gonadotropin stimulated testosterone production by porcine Leydig cells in culture

Pretreatment of primary porcine Leydig cell cultures with human leukocyte interferon suppressed the subsequent hCG-stimulated testosterone production in a dose-dependent manner, with an ED50 at 13 IU/ml. The treatment had no effect on hCG-binding to its receptor, and the inhibition of testosterone production was not abolished by 8Br-cAMP addition. The results indicate that the site of interferon action on hCG-stimulated testosterone production in primary cultures of porcine Leydig cells is located distal to cAMP formation.     

Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture.

Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process. In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome. After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml. At 11 days in culture, samples were removed for morphological characterization and determination of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD). Testosterone concentration was determined by RIA in the culture medium at different intervals. Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions. No mature Leydig cells were present. In basal conditions, the percentages of 3 beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%, respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng.10(6) cells.24 h, respectively. In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng.10(6) cells, respectively). Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: changes with age and effects of cryptorchidism

The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P < 0.05) from prepubertal age (4-6 mo) to pubertal age (6-12 mo), and then declined (P < 0.05) from pubertal age to post-pubertal age (1-5 y), reaching a plateau. Plasma testosterone concentrations increased (P < 0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P < 0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05-0.43 ng/ml) and testosterone (range: 0.10-0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL3 < 0.02 ng/ml; testosterone < 0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.     

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.     

Effects of testosterone, estradiol, aromatase inhibitor, gonadotropin and prolactin on the response of mouse testes to acute gonadotropin stimulation

These studies determined the local acute responsiveness of the testis to intratesticular administration of human chorionic gonadotropin (hCG) under basal, stimulated (systemic hCG pre-treated), hypogonadotropic (steroid pre-treatment) and hyperprolactinemic conditions in male mice. In addition, testicular testosterone (T) levels were determined after intratesticular administration of the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) or progesterone under basal or hCG-stimulated conditions. Intratesticular administration of 0.025, 0.25, 2.5 or 25 mIU hCG resulted in a dose-dependent (3- to 14-fold) increase in testicular T concentrations in hCG compared to vehicle-injected testes. Systemic (i.p.) pre-treatment with 5 IU hCG 24 h before prevented any further increases in the already elevated (10-fold basal) T levels after direct intratesticular hCG injection. Pretreatment with 250 micrograms testosterone propionate (TP) reduced basal testicular T concentrations, but resulted in increased responsiveness to intratesticular hCG administration. In contrast, estradiol benzoate (EB) pretreatment, which also reduced basal testicular T concentrations, did not affect the testicular responsiveness to hCG. Hyperprolactinemia reduced testicular responsiveness to intratesticular administration of 0.025, 0.25 or 2.5 mIU hCG, but basal levels of testicular T were elevated. One hour after intratesticular injections of an aromatase inhibitor, 4-OHA; (0.25 micrograms) testis, T levels were increased in males pre-treated with 5 IU hCG (i.p.) 24 h earlier. Higher doses of 4-OHA (2.5, 25 or 250 micrograms) resulted in significant, dose-related increases in basal testicular T levels which were attenuated by hCG-pre-treatment. Intratesticular administration of 20 micrograms progesterone increased testicular T concentrations 2.7-fold, but this effect was attenuated (1.5-fold) in hCG-pre-treated mice, suggesting that enzymatic lesions beyond progesterone may be involved in hCG-induced testicular desensitization. These results indicate that testicular responsiveness to hCG depends on the existing levels of gonadotropic stimulation. However, it is evident that estrogens and prolactin also influence the sensitivity of the testis to gonadotropin.     

Ovarian response to human chorionic gonadotropin or gonadotropin releasing hormone in cats in natural or induced estrus

Human chorionic gonadotropin (hOG) or gonadotropin releasing hormone (GnRH) was given alone or with repeated coital stimuli to study ovarian activity and ovulation in the domestic cat. Adult cats in natural estrus (NE) or treated with follicle stimulating hormone (FSH-P) to induce estrus (2.0 mg/d for 5 d; IE) were assigned to one of five treatments: I, mating (M) only (three times daily for the first 3 d of estrus); II, M + hOG (250 IU, i.m. on Days 2 and 3 of estrus); III M + GnRH (25 mug, i.m. on Days 2 and 3 of estrus); IV, hOG only (250 IU, i.m. on Days 2 and 3 of estrus); or V, GnRH only (25 mug on Day 2 and 3 of estrus). Overall, IE females produced a greater (P < 0.05) number of corpora lutea (7.6 +/- 0.9) and unovulated follicles (18.9 +/- 2.1) than NE cats (4.9 +/- 0.6 and 3.6 +/- 0.9, respectively). For both NE and IE females, the M + hOG treatment (II) produced a greater number (P < 0.05) of ovulations (9.1 and 13.9, respectively) than any other ovulatory regimen (I, 4.1, 6.6; III, 4.1, 7.8; IV, 4.0, 6.2; V, 4.1, 5.6, respectively). These results indicate that 1) the excessive follicle number resulting from FSH-P treatment cannot be reduced with any of the hOG or GnRH treatments tested and 2) the use of hOG with copulatory stimuli synergistically enhances the ovulatory response of cats experiencing a natural estrus or those treated with FSH-P.     

Changes in plasma concentrations of insulin-like peptide 3 and testosterone from birth to pubertal age in beef bulls

The objectives were to: (1) develop an enzyme immunoassay (EIA) for insulin-like peptide 3 (INSL3) or relaxin-like factor (RLF) in bovine plasma; (2) investigate changes of plasma INSL3 concentrations from birth to pubertal age of beef bulls; and (3) compare changes in plasma concentrations of INSL3, testosterone, and LH. Plasma samples were collected from beef bull calves (n = 15) at birth (0 d) and at 28, 56, and 84 d after birth. Furthermore, in beef bulls around pubertal age (n = 26; age range 3 to 22 mo), plasma samples were collected at 1 to 4 mo intervals. Plasma INSL3 concentrations increased (P < 0.05) from 0 to 28, 28 to 56, and from 56 to 84 d of age. Plasma testosterone concentrations increased (P < 0.001) from 0 to 28 d, and from 28 to 56 d, but did not change from 56 to 84 d. For bulls around pubertal age, plasma INSL3 concentrations did not change from the prepubertal phase (3 to 6 mo) to the early pubertal phase (6 to 12 mo), but increased (P < 0.05) from the early to late pubertal phases (12 to 18 mo), and from the late pubertal to postpubertal phases (18 to 22 mo). Plasma testosterone concentrations increased from the prepubertal to early pubertal phases (P < 0.001), but did not change thereafter. Plasma LH concentrations did not change from 0 d to 84 d, but decreased (P < 0.001) from prepubertal to early pubertal phase, with no significant change thereafter. Plasma INSL3 concentrations increased during the first 3 mo of life and throughout the pubertal age in beef bulls. There were similar dynamic patterns for INSL3 and testosterone during the first 3 mo of life, but patterns subsequently diverged in bulls around pubertal ages.     

Changes in plasma concentrations of insulin-like peptide 3 and testosterone from birth to pubertal age in beef bulls

The objectives were to: (1) develop an enzyme immunoassay (EIA) for insulin-like peptide 3 (INSL3) or relaxin-like factor (RLF) in bovine plasma; (2) investigate changes of plasma INSL3 concentrations from birth to pubertal age of beef bulls; and (3) compare changes in plasma concentrations of INSL3, testosterone, and LH. Plasma samples were collected from beef bull calves (n = 15) at birth (0 d) and at 28, 56, and 84 d after birth. Furthermore, in beef bulls around pubertal age (n = 26; age range 3 to 22 mo), plasma samples were collected at 1 to 4 mo intervals. Plasma INSL3 concentrations increased (P < 0.05) from 0 to 28, 28 to 56, and from 56 to 84 d of age. Plasma testosterone concentrations increased (P < 0.001) from 0 to 28 d, and from 28 to 56 d, but did not change from 56 to 84 d. For bulls around pubertal age, plasma INSL3 concentrations did not change from the prepubertal phase (3 to 6 mo) to the early pubertal phase (6 to 12 mo), but increased (P < 0.05) from the early to late pubertal phases (12 to 18 mo), and from the late pubertal to postpubertal phases (18 to 22 mo). Plasma testosterone concentrations increased from the prepubertal to early pubertal phases (P < 0.001), but did not change thereafter. Plasma LH concentrations did not change from 0 d to 84 d, but decreased (P < 0.001) from prepubertal to early pubertal phase, with no significant change thereafter. Plasma INSL3 concentrations increased during the first 3 mo of life and throughout the pubertal age in beef bulls. There were similar dynamic patterns for INSL3 and testosterone during the first 3 mo of life, but patterns subsequently diverged in bulls around pubertal ages.     

Cytology of the interstitial tissue in scrotal and abdominal testes of post-puberal boars

The interstitial tissue of the testes from healthy boars, and unilateral and bilateral abdominal cryptorchid boars was examined by light and transmission electron microscopy. The left and right testes of healthy boars, and the left (scrotal) testis of unilateral cryptorchid boars had abundant mature Leydig cells, few fibroblasts and mast cells, scarce and small blood vessels, and little lymphatic areas. The right (abdominal) testis of unilateral cryptorchid boars contained abundant Leydig cells, fibroblasts and erythrocytes, scarce mast cells, and frequent blood vessels; Leydig cells exhibited either a mature but degenerative appearance or an immature appearance, and fibroblasts displayed immaturity signs. The interstitial tissue of the left (abdominal) testes of bilateral cryptorchid boars had small blood vessels surrounded by erythrocytes, lymphocytes, and few plasma cells, and abundant mature and immature Leydig cells, immature fibroblasts, and mast cells. Mature Leydig cells showed mid or advanced degeneration, and immature Leydig cells displayed either non-degenerative or degenerative patterns. The right (abdominal) testes of bilateral cryptorchid boars contained scarce immature Leydig cells in advanced degeneration, large fibrous and adipose areas, and blood vessels. These results indicated that unilateral abdominal cryptorchidism affect neither the structural nor the cytologic features of the interstitial tissue in scrotal testes. Unilateral and bilateral cryptorchidism induced abnormal differentiation of Leydig cells and fibroblasts leading to decreased steroid production and increased collagenization in abdominal testes.     

Effect of human chorionic gonadotropin administration on pituitary and luteal response to gonadotropin-releasing hormone

The effect of human chorionic gonadotropin (hCG) administration on the pituitary and luteal responses to acute gonadotropin-releasing hormone (GnRH) administration at the mid luteal phase (LP) were studied in 20 infertile women. Patients were divided into 2 groups. In 1 group (n = 8), hCG (5,000 IU i.m.) was injected in a single shot on day 5 of LP. Sixty hours later (day 8 of LP) blood samples were taken every 15 min for 180 min; then 25 micrograms GnRH were acutely administered intravenously and blood samples taken at 185, 195, 210, 225, 240, 255, 270, 285 and 300 min. In the other 12 patients the same experimental design with GnRH was performed on day 8 of an untreated LP. Plasma LH, FSH, beta-hCG, progesterone and estradiol (E2) were assayed. The responsiveness of different hormones to GnRH was evaluated as integrated secretory area for 120 min after injection (sISA) and as the absolute increase with respect to the area under basal conditions before a GnRH administration (bISA). hCG-treated patients showed higher basal and bISA plasma values of LH/hCG than controls (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody recognition of a human chorionic gonadotropin epitope (hCGbeta66-80) depends on local structure retained in the free peptide

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical β3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGβ(66-80) and the closely related luteinizing hormone (LH) fragment LHβ(86-100), which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.