Luteinizing Hormone/human Chorionic Gonadotropin Research Articles

Effect of estradiol and progesterone on myometrial LH/hCG receptors in pigs.

High affinity luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors have been identified in porcine, rabbit and rat uteri and immunocytochemically demonstrated in the human uterus. We have now assessed the effect of estradiol and progesterone on the capacity and affinity of LH/hCG binding sites in crude membrane fractions of porcine myometria. Nineteen cross-bred gilts were ovariectomized at 6-7 months of age. Five weeks later, the experiment was conducted and gilts were given estradiol benzoate 2 mg (N = 5), progesterone 50 mg (N = 4) and 2 mg of estradiol benzoate plus 50 mg of progesterone in 2 ml of corn oil (N = 6), im for five consecutive days. Controls (N = 4) received 2 ml of the vehicle. Gilts were hysterectomized 24 h after the last injection. Blood samples for assays of LH, estradiol and progesterone were collected 1 h before hysterectomy. The numbers and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. The results indicate that treatment with estradiol benzoate increases (p less than 0.01) the number of LH/hCG binding sites compared with gilts receiving corn oil. Progesterone treatment caused elevation in the number of LH/hCG receptors (p less than 0.05), when compared with estradiol alone (2.9 +/- 0.3 vs 1.2 +/- 0.1 fmol/mg protein, respectively). Combined administration of estradiol and progesterone increased receptor capacity to 2.7 +/- 0.4. Steroid treatment did not alter the affinity (Ka) of [125I]hCG binding to receptors and it varied from 1.8 +/- 0.8 to 2.9 +/- 0.2 x 10(11) l/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmolytes improve the reconstitution of luteinizing hormone/ human chorionic gonadotropin receptors into proteoliposomes

Rat ovarian membrane luteinizing hormone/ human chorionic gonadotropin (LH/hCG) receptor was reconstituted into proteoliposomes. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/detergent ratio. Trypsinization of the LH/hCG receptor containing proteoliposomes indicated that approximately 57% of hCG binding sites were oriented extravesicularly. The presence of 20% glycerol or other osmolytes during reconstitution increased the accessibility of LH/hCG receptors but not the activity of adenylate cyclase in proteoliposomes. This beneficial effect was independent of any specific detergent or its presence during detergent solubilization of proteins. Dynamic properties of membranes were monitored by electron spin resonance of 16-, 12-, and 5-doxyl stearic acid. Reconstituted proteoliposomes contain less ordered membrane lipids than do native membranes. Addition of glycerol before reconstitution increased the order of lipid bilayer and shifted it to the physical state of the native membrane. These findings are consistent with the hypothesis that a rise in membrane ordering increases the accessibility of membrane-bound LH/hCG receptors.

Hormonal regulation of gonadotropin receptor mRNA in rat ovary during follicular growth and luteinization

To investigate the regulation of the follicle-stimulating hormone (FSH) and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor genes by gonadotropins, we examined the effect of pregnant mare's serum gonadotropin (PMSG) or PMSG-hCG on the expression of FSH and LH/hCG receptors in rat ovaries. After administration of PMSG, Northern blot analysis using the FSH receptor cDNA probe revealed that a major band of 2400 nucleotides was detected which reached the maximal level on day 3. On the other hand, the level of LH/hCG receptor mRNA, a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300 and 1200 nucleotides, increased progressively during 4 days. Treatment with hCG resulted in a decrease of FSH and LH/hCG receptor mRNA levels, and the level of FSH receptor mRNA was completely suppressed. Although the level of LH/hCG receptor mRNA was also suppressed from 3 h to an almost undetectable level at 24 h after hCG injection, it recovered to the control level by 48 h and exceeded this level several fold by 72 h. The reappearance of LH/hCG receptors following desensitization was preceded by an increase in mRNA levels. These studies demonstrate that hormonal regulation of gonadotropin receptor mRNAs on rat ovary reflects the changes in gonadotropin receptor levels.

Effects of human chorionic gonadotrophin on contractile activity of steroid-primed pig myometrium in vitro

We examined the effects of (a) oestrogen and progesterone on concentrations of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in uterine smooth muscle in vivo and (b) hCG on spontaneous myometrial contractions in vitro. Ovariectomized gilts received 2 ml corn oil (control; n = 5), 2 mg oestradiol benzoate (n = 6) or 20 mg progesterone (n = 5) for 5 days. Gilts were hysterectomized 8 h after the last injection and longitudinal sections of myometrium were incubated in modified Krebs' solution with 0 or 10 i.u. of hCG (n = 10/gilt) for 4 h at 37 degrees C in 95% O2:5% CO2. After incubation, myometrial sections were placed in a tissue chamber perfused with Krebs' solution and mechanical activity was recorded for 30 min. Cell membrane fractions were prepared from myometrial tissue not used for in-vitro studies and analysed for LH/hCG receptors. Treatment with oestradiol benzoate increased (P less than 0.01) the number of LH/hCG-binding sites compared with gilts receiving corn oil or progesterone. Incubation of myometrium with hCG reduced (P less than 0.01) the frequency and amplitude of spontaneous uterine contractions in gilts treated with oestradiol benzoate. In contrast, hCG had no effect (P greater than 0.05) on the pattern of myometrial contractions in gilts given corn oil or progesterone. These results indicate that oestradiol promotes the synthesis of LH/hCG receptors in pig myometrium and incubation of oestrogen-primed tissue with hCG has a quiescent effect on myometrial contractility.

Inhibition of luteinizing hormone-human chorionic gonadotropin binding by retinoids in a Leydig cell line

Treatment of K9 mouse Leydig cells with 3×10 −6 M retinol (R) and retinoic acid (RA) resulted in 75% and 65% reduction of 125I-labeled hCG binding respectively, when assayed at 35° C. This effect was dose-dependent and was first detected 12 h after initiation of treatment: it was maximal at 48 h for RA. R and RA had no significant effect on the rate of internalization and degradation of 125I-hCG as measured by disappearance of acid-releasable (i.e. surface-bound) radioactivity from the cells and by the appearance of trichloracetic acid-soluble label in the medium. When exposed to increasing concentrations of hCG for 24 h, both retinoid-treated and control cells ‘down-regulated’ their gonadotropin receptors with the same dose-dependent pattern. The kinetics of reappearance of the receptors was similar for retinoid-treated and control cells, but for treated cells the maximal number of receptors reinitiated at 24 h never exceeded 40% of the values observed with control cells. Scatchard plot analysis confirmed a decrease in hCG receptor number from ∼26,000 to ∼6400 and ∼3500 sites per cell after R and RA treatment, K d values for 125I-hCG binding were 2×10 −10 M, 7.3×10 −11 M and 6.9×10 −11 M for control, R- and RA-treated cells respectively. On the basis of our data it is likely that retinoid-induced reduction in 125I-hCG binding to K9 Leydig cells is due to decreased receptor synthesis.

Luteinizing hormone and human chorionic gonadotropin receptors in human corpora lutea from clomiphene citrate-induced cycles

Midluteal phase corpora lutea (CL) obtained from women induced with 50 mg (n = 5), 100 mg (n = 5), and 150 mg (n = 5) of clomiphene citrate (CC) were measured for luteinizing hormone/human chorionic gonadotropin (LH/hCG) concentrations and cytosol progesterone (P) and 17 α -hydroxyprogesterone (17-OHP) and compared with midluteal phase CL from eight normal women (controls). More CL (26) that were significantly heavier (2.0 ± 0.3 g, [mean ± SEM]) were obtained with CC than in controls (10). Clomiphene citrate treatment increased LH/hCG receptor concentrations and the dissociation constant significantly from 69 ± 12 fmol/mg protein and 1.1 ± 0.2 × 10 − 10 M, respectively, in controls to 112 ± 6 fmol/mg protein and 2.1 ± 0.1 × 10 − 10 M. Cytosol P and 17-OHP levels were not significantly increased. Cumulatively these cellular effects may be responsible for increasing serum P and responsiveness to hCG and for correcting luteal dysfunction.

Cytosol progesterone and 17α-hydroxyprogesterone levels and luteinizing hormone and chorionic gonadotropin receptors in human corpora lutea

Cytosol progesterone (P) and 17 alpha-hydroxyprogesterone (17-OHP) levels and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors were measured in 27 corpora lutea and four corpora albicantia. Cytosol P concentrations were highest in corpora lutea (mean +/- SEM, 3.1 +/- 0.8 micrograms/g) during the midluteal phase (days 15 to 19) rather than the early (2.2 +/- 0.8 micrograms/g, days 20 to 25) and late luteal phases (1.8 +/- 0.8 micrograms/g, days 26 to 30). Cytosol 17-OHP concentrations also were 3.3 +/- 0.5, 4.3 +/- 0.6, and 3.3 +/- 1.0 micrograms/g in early, midluteal, and late luteal phases, respectively, and was significantly inversely correlated with occupied LH/hCG receptors in midluteal phase. Corpora albicantia had the lowest P (0.3 +/- 0.05 microgram/g) and 17-OHP (0.9 +/- 0.6 micrograms/g) concentrations. Cytosol P and 17-OHP may therefore reflect the balance between the luteal cell production and secretion, whereas the amount of occupied and unoccupied LH/hCG receptors may partially explain the relationship between LH and P secretion.

Ontogeny of the gonadal receptor for luteinizing hormone in the pig

Homogenates of porcine ovaries and testes collected between 70 d post coitum and 42 d post partum were incubated with radiolabelled human chorionic gonadotropin (hCG) to determine the presence and relative amounts of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors. Specific binding of 125I-hCG to ovaries and testes occurred at all stages of fetal and postnatal development. Ovarian tissue possessed relatively low affinity, high capacity LH/hCG binding sites that were most numerous at Day 80 of gestation and decreased thereafter. In contrast, high affinity, low capacity LH/hCG binding sites were found in the testes. In males, the total number of LH/hCG binding sites remained stable until near term and then increased with age, but the number of sites per gram of testicular tissue did not change (P>0.05). In summary, differential binding of LH/hCG in gonadal tissue occurred in male and female piglets during pre- and post-natal periods, and this binding reflected the known differential pattern of development of the male and female gonad.

Gonadotropin receptor occupancy and stimulation of cAMP and testosterone production by purified Leydig cells: Critical dependence on cell concentration

Rat testicular interstitial cells have been separated by discontinuous/continuous gradient of Percoll, yielding four cell fractions. The light cells in fraction I bound luteinizing hormone/human chorionic gonadotropin (LH/hCG) with high affinity but were not steroidogenic in response to hormone. Fraction II consisted mainly of germ cells. Although fraction III contained Leydig cells, this fraction was contaminated with germ cells and was less responsive to hormone as compared to the Leydig cells in fraction IV. The Leydig cells in fraction IV produced cAMP and testosterone in response to hormone action in a manner which was critically dependent upon cell concentration. The production of cyclic adenosine monophosphate (cAMP) in the presence of saturating concentrations of hCG (2.4 × 10 t1̄0 M) was linear as a function of cell concentration up to 7.0 × 10 6 cells/1.25 ml and thereafter, a slight inhibition (26%) was seen at 10 × 10 6 cells/1.25 ml. The average value for cAMP production by hCG was 133.8 ± 8.5 pmol cAMP/2 × 10 6 cells. The production of testosterone was biphasic, increasing linearly up to 5 X 10 6 cells/1.25 ml and decreasing thereafter. Two million cells, in the presence of 2.4 × 10 −10 M hCG, produced an average of 24.2 ± 1.7 ng of testosterone in reaction volumes ranging from 1 to 2 ml whereas the same number of cells only produced 5.1 ± 0.6 ng of testosterone in 250 μ1. The binding of 125I-labeled hCG to the same batch of cells increased with increasing cell concentrations as expected but under the conditions of maximal steroidogenesis at low cell concentrations (1.25 2.0, and 2.5 × 10 6 cells/1.25 ml), it was barely detectable. Thus, we conclude that there is an inverse relationship between the parameters of binding and biological response in purified Leydig cells.

Cyclic AMP-dependent and -independent Regulation of Cholesterol Side Chain Cleavage Cytochrome P-450 (P-450scc) in Rat Ovarian Granulosa Cells and Corpora Lutea.: cDNA and deduced amino acid sequence of rat P-450scc

Abstract We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.

Effects of interleukins 1, 2 and 3 on follicle-stimulating hormone-induced differentiation of rat granulosa cells

A growing body of evidence indicates that substances released by activated immune cells can directly influence the functions of various endocrine cells. In the present study, the direct in vitro effects of interleukins (IL) 1, 2, and 3 on follicle-stimulating hormone (FSH)-stimulated Steroidogenesis and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction in granulosa cells were examined. In the absence of FSH, none of the interleukins stimulated steroid production or LH/hCG receptor induction during a 2-day culture period. However, in the presence of FSH, both IL-1α and IL-1β inhibited, in a dose-dependent manner, progesterone and estrogen production as well as LH/hCG receptor induction in response to FSH. In contrast, both agents augmented 20α-hydroxy-4-pregnen-3-one (20α-OH-P) production stimulated by FSH. In all cases, less IL-1β than IL-1α was required to produce a comparable effect. IL-2 slightly, but significantly, enhanced both FSH-stimulated progesterone and 20α-OH-P production but had no effect on FSH-stimulated estrogen production or LH/hCG receptor induction. IL-3 potentiated the 20α-OH-P response to FSH by up to 65% but had no effect on FSH-stimulated progesterone or estrogen production or LH/hCG receptor induction. These data suggest that the interleukins, which are key mediators of immune responses, may affect mechanisms crucial for the maturation and differentiation of granulosa cells and thus may also play a regulatory role in reproductive function.

Synergistic effect of insulin and follicle-stimulating hormone on biochemical and morphological differentiation of porcine granulosa cells in vitro.

Insulin and follicle-stimulating hormone (FSH) have been shown to facilitate granulosa cell differentiation in vitro. To gain insight into this process, we evaluated the effects of these hormones, alone and in combination, upon the biochemical parameters of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction and progesterone secretion concomitantly with morphometric analysis of granulosa cell ultrastructure and LH/hCG receptor distribution by quantitative autoradiography under light microscopy. Granulosa cells isolated from small antral follicles (controls) cultured in the absence of exogenous hormones exhibited few microvilli and gap junctions; the mitochondria, endoplasmic reticulum, and Golgi complex were all poorly developed. Progesterone secretion was negligible and the cells bound little [125I]iodo-hCG. Insulin treatment increased gap junction formation, and the extent of smooth and rough endoplasmic reticulum and Golgi complex development (all p less than 0.05) but did not affect mitochondrial ultrastructure or volume. Insulin treatment modestly but significantly increased [125I]iodo-hCG binding and progesterone secretion relative to controls (p less than 0.001). FSH treatment had a similar effect to insulin on cell ultrastructure and additionally enhanced development of the mitochondria and smooth endoplasmic reticulum as well as formation of the microvilli (p less than 0.05). FSH significantly increased [125I]iodo-hCG binding and progesterone secretion relative to insulin-treated samples (p less than 0.001). Combined treatment with insulin and FSH markedly increased gap junction and microvilli formation and enhanced the development of the smooth endoplasmic reticulum and the Golgi complex relative to treatment with either hormone alone (p less than 0.05). Additionally, the combined treatment produced larger mitochondria with tubular christae. Consistent with the morphological development, the combined treatment of insulin and FSH significantly increased progesterone secretion and [125I]iodo-hCG binding (p less than 0.001). Autoradiographic analysis showed that aggregated cells in general exhibited higher LH/hCG receptor density than nonaggregated cells, and a significantly higher overall receptor density compared to nonaggregated cells or to cells treated either with insulin or FSH alone. Our results indicate that insulin and FSH facilitate morphological differentiation of the granulosa cell in a synergistic manner, stimulating gap junctions and microvilli formation and enhancing development of the mitochondria, endoplasmic reticulum, and Golgi complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of receptor-mediated gonadotropin action in rat testes by dietary fat.

The effect of feeding diets enriched with 18:2 omega 6, 18:3 omega 3, or saturated fatty acids on lipid composition and receptor-mediated action of luteinizing hormone/human chorionic gonadotropin (LH/hCG) in rat testicular plasma membranes was investigated. Linoleic and alpha-linolenic acid treatments reduced total phospholipid and cholesterol content of the testicular plasma membrane and altered membrane phospholipid composition. Change in phospholipid and cholesterol content after feeding the polyunsaturated fats decreased cholesterol to phospholipid ratios and binding capacity of the LH/hCG receptor in the testicular plasma membrane. LH-stimulated adenylate cyclase activity was decreased in animals fed the linolenic acid-rich diet. NaF-stimulated adenylate cyclase activity was decreased in animals fed diets high in either polyunsaturated fatty acid. Decreased plasma membrane LH/hCG receptor content was associated with decreased testosterone production in Leydig cells in response to LH in the linolenic acid-fed group. It is suggested that change in cholesterol-to-phospholipid ratios alters the physical properties of testicular plasma membranes in a manner that influences accessibility of LH/hCG receptors in testicular tissue.

Dynamics of bioactive luteinizing hormone-human chorionic gonadotropin during the first 7 days of life.

Serum luteinizing hormone-human chorionic gonadotropin bioactivity (B-LH) was measured daily in seven male and four female full-term newborns during the first 7 days of life. The B-LH levels were elevated in both sexes during the 1st day of life; subsequently, values decreased in both sexes. In males, they reached a nadir on the 4th day of life. A gradual secondary rise was then observed with B-LH levels on the 7th day significantly higher than on day 4 (p less than 0.025). By contrast, the B-LH levels in the females continued a gradual decline to levels significantly lower on day 7 as compared to day 4 (p less than 0.05). To determine whether pulsatile B-LH secretion occurs in newborns, serum concentrations were measured every 20 min for 2 h in eight male and seven female full-term neonates on the 7th day of life. Pulsatile secretion of B-LH was detected in six males and six females. This study demonstrates that pulsatility of gonadotropin secretion is characteristic of neonates as early as 7 days of life and that there is a dichotomy between the levels of B-LH in males and females; levels in females decline progressively from day 1 through 7, whereas in males, a nadir is reached on day 4 with a secondary rise developing thereafter. This male sex-specific rise is presumably the drive responsible for the characteristic postnatal increase in testosterone which peaks at 1 to 2 months of age.

Luteinizing Hormone Receptor Number and Affinity in Corpora Lutea from Prepuberal Gilts Induced to Ovulate and Spontaneous Corpora Lutea of Mature Gilts

This study was designed to determine if luteal cell receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) contribute to the previously demonstrated abnormal function of induced corpora lutea (CL) in gilts. Twenty-five prepuberal (P) gilts, induced to ovulate with 1,500 IU pregnant mare serum gonadotropin followed 72 h later with 500 IU hCG (d 0 = day of hCG), and 22 mature (M) gilts that had displayed two or more estrous cycles were ovariectomized (OVX) on d 10, 14, 18, 22 or 26 after the onset of estrus. All gilts except those OVX on d 10 were hysterectomized between d 6 and 9 to ensure luteal maintenance. The CL were stored at -196 degrees C until determination of LH/hCG receptor number and dissociation constant (KD) by saturation analysis. Receptor number was greater for M than for P gilts on d 14 (P less than .07) and d 18 (P less than .01). The KD was greater in M than in P gilts on d 14 (P less than .01) and d 18 (P less than .0001). The LH/hCG receptor number and KD of P gilts remained the same throughout the days studied. The LH/hCG receptor number (fmol/mg protein) of M gilts was elevated on d 10, 14, and 18 (50.8, 50.4 and 51.4, respectively) and decreased on d 22 (26.5) and d 26 (25.4) to values similar to those of P gilts. In M gilts, KD increased on d 14, remained high on d 18 and decreased on d 22. We suggest that abnormal function of induced CL in P gilts may be due to an elevated LH receptor number.

Antigonadal activity of the neurohypophysial hormones: in vivo regulation of testicular function of hypophysectomized rats.

The "antigonadal" potential of the neurohypophysial hormones, previously demonstrated in vitro, was evaluated in vivo using hypophysectomized male rats. This approach minimizes the likelihood that the in vivo "antigonadal" effect of the neurohypophysial hormones may be due to their ability to attenuate the release of pituitary gonadotropins. Given that the identity of the putative endogenous occupant of testicular pressor-selective neurohypophysial receptors remains uncertain, use was made of a substitute probe, arginine vasotocin (AVT), the utility of which has been demonstrated in vitro. Concurrent in vivo treatment of follicle-stimulating hormone (FSH; 5 micrograms/rat/day)-maintained immature hypophysectomized rats with increasing doses of AVT (0.25-25 microgram/rat/day) produced significant (P less than 0.05) dose-dependent inhibition of the testicular luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor binding capacity (but not affinity; Kd = 1.8 X 10(-10) M) from 8.8 +/- (standard error; SE) 0.4 ng/testis to a level (3.2 +/- 0.2 ng/testis) lower than that of controls (64% reduction). This AVT-induced decrease in the testicular LH/hCG receptor content of FSH-maintained immature hypophysectomized rats was associated with significant (P less than 0.05) decrements in the hCG- and N6, 2'-O-dibutyryladeosine cyclic 3',5'-monophosphate [( Bu]2cAMP)-stimulated accumulation of 3 alpha-hydroxy-5 alpha-androstan-17-one (androsterone; 52% and 42% inhibition, respectively), with virtual elimination (98% inhibition) of the forskolin-stimulated accumulation of extracellular cAMP by testicular incubates in vitro, as well as with profound suppression of spermatogenesis. Taken together, these observations indicate that the "antigonadal" effect of the neurohypophysial hormones previously demonstrated in vitro, can be fully reproduced in vivo, and that the "antigonadal" activity of the neurohypophysial hormones may be accounted for, in large part, by decreased testicular LH/hCG binding capacity, stimulable adenylate cyclase activity, and cAMP-supported androgen biosynthesis.

Use of individual human follicles to compare oocyte in vitro fertilization to granulosa cell in vitro luteinization

Granulosa-lutein (G-L) cells from individual follicles aspirated during cycles of in vitro fertilization-embryo transfer were examined after 3 and 6 days in culture. G-L cells from follicles that contained an oocyte that fertilized in vitro were compared with G-L cells from follicles that contained an oocyte that did not fertilize in vitro. Spent culture media was assayed for progesterone at days 3 and 6 of culture and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor content of G-L cells was determined at day 6. G-L cell cultures from follicles that contained an oocyte that fertilized in vitro produced significantly more progesterone over 3 and 6 days of culture than those obtained from follicles in which the oocyte did not fertilize. Furthermore, LH/hCG receptor content after 6 days was significantly higher in G-L cells obtained from follicles with fertilized oocytes compared with follicles with unfertilized oocytes. Increased progesterone output and LH/hCG receptor acquisition demonstrate more maturation or "luteinization" by G-L cells aspirated from individual follicles that contain oocytes that fertilized in vitro.

Subunits of luteinizing hormone-human chorionic gonadotropin receptor from bovine corpora lutea.

A batch of 24 mg of luteinizing hormone-human chorionic gonadotropin (LH-hCG) receptor was isolated from bovine corpora lutea. The LH-hCG receptor showed specific binding with hCG. The receptor-hCG complex activated the regulatory Ns protein isolated from rabbit liver, which in turn stimulated adenylate cyclase to convert ATP into cAMP in vitro, attesting to the biological activity of the purified LH-hCG receptor. The LH-hCG receptor was treated with 2% sodium dodecyl sulfate (SDS) to prepare the molecular weight (Mr) 280K dimer and with 50 mM dithiothreitol (DTT) to prepare the Mr 120K monomer and subunits of Mr 85K and 38K. Oligomers of various molecular weights were recovered from gel filtration columns due to the reassociation of disulfide bonds between monomers and subunits. Hence, the receptor monomer was also dissociated into subunits of Mr 85K and 38K by reduction of -S-S-bonds with 50 mM DTT in 2% SDS and alkylation of sulfhydryl groups in the presence of 100 mM N-ethyl-maleimide. The subunits were separated by gel filtration through columns of Ultrogel AcA-44 and Sephadex G-75. The yields of the purified alkylated subunits of Mr 85K and 38K were 1.8 and 1.5 mg, respectively. Each subunit migrated as a single entity in SDS-polyacrylamide gel electrophoresis. The monomer of the receptor of Mr 120K showed specific binding with 125I-hCG, suggesting it to be the minimum molecular weight functional unit of the receptor. The Mr 85K and 38K subunits bound 125I-hCG, which could not be displaced with unlabeled hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical visualization of luteinizing hormone-human chorionic gonadotropin (LH-hCG) receptors in Leydig cells in primary culture.

Visualization of human chorionic gonadotropin (hCG) binding sites was obtained by immunocytochemical reaction on Leydig cells cultured in chemically defined medium. After a short in vitro incubation with hCG or luteinizing hormone (LH), the cells were fixed and the bound molecules were revealed using anti-hCG or anti-LH antisera. In both cases the immunocytochemical reaction appeared as granulations at the cell surface. After prolonged (48 hr) culture in the presence of 0.5, 5, or 50 ng/ml of hCG, the hormone receptor complex is still visible. Similarly, following short exposure to hCG (0.5 or 50 ng/ml) and one or two days of culture without hCG, the immunocytochemical reaction is still present. These observations suggest that the half-life of the bound hormone is very long. This in vitro system in which the amount and time of hormone exposure are precisely defined provides arguments in favor of a long-term maintenance of the receptor complexes at the cell surface.

Characterization of two steroidogenic cell types in the ovine corpus luteum.

A procedure which utilized elutriation was developed which permitted the separation of two populations of luteal cells from enzymatically dispersed corpora lutea taken from superovulated ewes on the 10-12th day of the estrous cycle. One population consisted of cells which measured 23-35 pm in diameter, while the other consisted of cells with a diameter of 12-22 pm. Preparations of both cell types were analyzed for rate of secretion of progesterone, responsiveness to secretagogues, and number of specific binding sites for luteinizing hormone/human chorionic gonadotropin (LH/hCG), and prostaglandins E2 and F2a (PGE2 and PGF2a). Without stimulation, the secretion of progesterone by large cells was approximately 20-fold greater than for an equal number of small luteal cells. However, the ability of large luteal cells to respond to LH (100 ng/mI) or N6,02-dibutyryladenosine 3’: 5’-cyclic monophosphoric acid (dbcAMP, 10 mM) was minimal and approximately 10% of that obtained with small luteal cells. Marked differences were also observed between the two cell types with respect to the number of specific binding sites for LH/hCG, PGE2 and PGF2 a. The large cells had very few receptors for LH/hCG compared to small cells (3074 receptors/large cell vs. 33,260/small cell during the breeding season). The majority of receptors for prostaglandins was found in large cells (68,143 sites of PGF2a and 10,955 sites for PGE2/large cells; 2115 sites for PGF2a and 904 sites for PGE2/small cell). These data suggest that functional differences exist between the large and small steroidogenic cells in the corpus luteum of the ewe. The large luteal cells appear to secrete most of the progesterone produced by the corpus luteum, however, this secretion appears to be independent of LH and dbcAMP. In contrast, the small luteal cells secrete minimal quantities of progesterone in the unstimnulated state butresponddramatically to LH or dbcAMP.