Luteinizing Hormone Preparations Research Articles

Luteinizing hormone (LH) formed a complex with an immunoglobulin G caused abnormally high levels of LH: A case report

We encountered a 30-year-old woman with remarkably elevated luteinizing hormone (LH) levels, as measured by electrochemiluminescent immunoassay (ECLIA), and no specific symptoms. We performed the following investigations: dilution linearity test, polyethylene glycol (PEG) precipitation test, immunoprecipitation test, protein G addition test, and high-performance liquid chromatography (HPLC) analysis. The linearity of patient’s serum was similar to that of a standard LH preparation, and non-specific reactions were not observed. The recovery rate of LH shown by the PEG precipitation test, immunoprecipitation test, and protein G addition test was low. Moreover, an abnormal peak in HPLC was located at a slightly larger molecular weight position than that of IgG. These results showed the presence of macro-LH, LH, and anti-LH-IgG autoantibody complex and suggested that the clearance of LH from the blood was delayed due to IgG binding, and therefore, the LH value was falsely high. We should keep the possibility of macro-LH in mind in cases of unexpectedly high LH values.

Use of SP‐Sephadexto Fractionate and Obtain Semi‐Pure Preparation from Whole Pituitaries of Indian Water Buffaloes (Bubalus bubalis)

Different charge isoforms of native Luteinizing hormone (LH) (dimeric form) V e/V o = 1.49 can be fractionated on SP‐Sephadex into four different charge isoforms (LHUB, LH25, LH50, and LH100) by stepwise elution using different molarities of Na2HPO4. LHUB was found to be difficult to purify, whereas LH50 and LH100 were found to be pure and highly immunoreactive against anti‐bLHβ serum as indicated by the results obtained from direct binding ELISA and Western blot analysis. SDS–PAGE of LH50 and LH100 showed two bands corresponding to the two subunits of LH. LH25 can be purified to homogeneity by rechromatography on S‐300. Purification of buLH as a highly immunoreactive preparation has also been described using SP‐Sephadex. This LH preparation (SP25B), which was obtained after slight modification in the pre‐existing protocol, has been found to be highly immunoreactive against anti‐bLHβ serum in direct binding ELISA. Being a very simple and reproducible method, it can be used to obtain pure LH preparation, as a reference material, in a short period of time for various immunoassays and bioassays.

Recombinant human luteinizing hormone: a partial physicochemical, biological and immunological characterization.

The aim of this study was to partially characterize the glycoform composition of a recombinant human luteinizing hormone preparation (rhLH; Serono), an early version of the material (LHadi) which is currently being assessed for clinical application. Specifically, the charge (pl) and internal carbohydrate complexity of this rhLH was examined and compared with that of an alternative commercially available form of recombinant LH (Crystal Chem) and a pituitary International Reference Preparation (IRP). All preparations were separated by charge by chromatofocusing them on a pH gradient (7-4) using a 4 ml mono-P column is conjunction with a fast performance liquid chromatography system and by complexity of the oligosaccharide structures using concanavalin A (con-A) lectin affinity chromatography. LH in both the unfractionated and fractionated material was assessed by immunoradiometric assay (IRMA, I-LH) and by the in-vitro Leydig cell bioassay (B-LH). Both assays were calibrated against IRP 80/552. The in-vitro biopotency of the preparations was 18187 (Serono rhLH), 12063 (Crystal Chem rhLH) and 6658 (80/552) IU/mg; biological:immunological ratios were 1.14 (80/552), 1.90 (Crystal Chem rhLH) and 1.99 (Serono rhLH). However, similar qualitative data were obtained by both bioassay and immunoradiometric assay following fractionation, with the median pl of the bioactive LH in the preparations being 5.5 (24% > pH 6), 5.52 (18% > pH 6) and 4.97 (0% > pH 6) for the Serono, Crystal Chem and pituitary preparations respectively. Further all three contain < 1% of the complex carbohydrate structures and between 36-44% and 56-63% of the intermediate and simple forms of bioactive LH. In conclusion, the Serono recombinant LH preparation has a higher in-vitro bioactivity and is more basic than the other two preparations although the complexity of its carbohydrate moities appears to be similar.

Gonadotrophin control of follicular function.

It has been known for over 50 years that both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are required to stimulate both follicular development and oestradiol synthesis. However, previous experiments employing FSH and LH preparations (whether of pituitary or urinary origin) have not been able to answer unequivocally, whether an observed response was solely due to either FSH or LH because they were not pure preparations. In view of the recent availability of both 100% pure recombinant human FSH and recombinant human LH, we now have a unique opportunity to test their contribution in the regulation of ovarian function. Such experiments may have important clinical implications as they offer a means to interpret the effect of 'pure' FSH preparations when used to stimulate ovarian function in women undergoing different therapeutic regimens. To test the contribution of LH to optimize ovarian responsiveness to FSH, 21-day-old hypophysectomized, immature, female rats were treated for a 2-day period with varying total doses of rec-FSH (30-72 IU and/or rec-LH at 12-hourly intervals. At 48 h after the first injection, ovaries were removed, weighed and used to isolate granulosa and thecal interstitial cells for assessment of basal and gonadotrophin-responsive steroidogenesis in vitro; homogenized to extract total RNA for Northern analysis of 17-hydroxylase/C17-20-lyase (cytochrome P-450C17); mRMA; or examined using in situ hybridization to determine the expression of P-450C17 in the rat graafian follicle. The experiments demonstrated the potential for rec-FSH to influence LH-responsive androgen synthesis (via a paracrine mechanism) which involves an up-regulation of thecal P-450C17 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of luteal regression, ovulation and development of new luteal tissue during early pregnancy in heifers

A study was conducted to determine whether luteal tissue formed after administration of exogenous luteinizing hormone (LH) was capable of maintaining pregnancy in the absence of the original corpus luteum (CL) of pregnancy in heifers. Nineteen beef heifers, confirmed pregnant by ultrasonography approximately 28 days after breeding, were randomly assigned to one of two groups. Nine heifers remained as untreated controls to determine the incidence of spontaneous embryonic or fetal loss. Each of the remaining ten heifers was given two Syncro-Mate-B (SMB) ear implants (12 mg of norgestomet) between 30 and 35 days after breeding (day of SMB implant was Day 1). An additional SMB was implanted on Day 8 and another on Day 15. All implants were removed on Day 20. On Day 2, SMB-implanted heifers were given an intramuscular (i.m.) injection of 500 μg of cloprostenol to induce luteal regression. On Day 5, regression of the CL was confirmed by ultrasonography and these heifers received a highly purified porcine LH preparation (50 mg Armour, i.m.); an additional 25 mg was injected i.m. on Day 6. Ovulation occurred in all ten LH-treated heifers and was detected on Day 7.2±0.1 (mean±SEM). In five of ten LH-treated heifers, the induced CL regressed, and was last detected by ultrasonography on Day 15.8±0.4 (range, Day 15–17). Pregnancy was not maintained in these heifers after SMB implants were removed. Pregnancy was maintained until Day 40 (end of experiment) in the remaining five LH-treated heifers and in all nine control heifers. In all five heifers in which the induced CL regressed, this CL had formed in the ovary contralateral to the original CL of pregnancy. In contrast, in four of five heifers in which the induced CL was maintained, this CL had formed in the ovary ipsilateral to the original CL of pregnancy. Although LH induced ovulation and CL formation in the absence of the CL of pregnancy and in the presence of continuous progestagen support, short-lived CL were formed in five of ten heifers.

Validation of an improved in vitro bioassay to measure LH in diverse species.

Cultured Leydig tumor cells (MA-10) respond to luteinizing hormone (LH) by synthesizing and secreting progesterone (P). The specificity of the response to LH prompted us to develop this system for use as a simple and rapid in vitro bioassay for LH. The aims of this study were to (1) improve sensitivity and reproducibility, and (2) optimize the assay for use in diverse animal species. A minimum sensitivity was observed at 0.05 mlU/well of LH with 0.5 x 10(4) cells/well for 1.5 hours. At higher concentrations of LH, shorter incubation periods also significantly stimulated P production. Addition of human LH standard or serum samples resulted in a dose-dependent increase in P production. Parallel dose-dependent curves were observed with LH preparations from mammalian, avian, and amphibian species. In conclusion, these findings demonstrate that (1) the assay is rapid, sensitive, and reproducible; (2) serum LH levels analyzed using this assay and the mouse Leydig cell bioassay are comparable; (3) shorter incubation times suggest the implementation of this assay for rapid qualitative determination of LH surges; and (4) the assay can be used for the analysis of samples from diverse species, especially those lacking radioimmunoassays. Therefore, this assay system allows for the simple and rapid measurement of circulating bioactive LH levels in humans and diverse animal species, and should provide insight regarding the role of bioactive LH in physiological and pathological conditions.

Full physiological maturation in vitro of immature mouse oocytes induced by sequential treatment with follicle-stimulating hormone and luteinizing hormone

Cumulus cell-enclosed immature mouse oocytes were matured in medium supplemented with various combinations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol. FSH or LH alone stimulated oocyte maturation, resulting in a significant increase in the rate of development to blastocysts following fertilization in vitro and embryo culture. There was no significant difference between FSH and LH. The effect of FSH was neutralized by FSH antiserum, while that of LH was not, indicating that the stimulation of maturation by LH was not due to FSH contamination in the LH preparation. When LH was added after 2 hr of culture with FSH (sequential combination), blastocyst development was significantly increased compared with FSH alone, reaching the same level as the in vivo matured oocytes. The addition of estradiol, 0.1 ng/ml, to the sequential combination of FSH and LH had no effect, while 0.01 and 1 ng/ml produced a negative effect. The birth rate of normal live offspring following embryo transfer showed no significant difference between embryos derived from oocytes matured in vivo and in vitro (sequential combination with or without 0.1 ng/ml estradiol) or between the two in vitro treatment groups.

Elephant pituitary gonadotropins

We describe for the first time the purification and some properties of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) isolated from anterior pituitary tissue of the African elephant ( Loxodonta africana). Methodology previously applied to equine and donkey pituitaries was used to obtain purified preparations of elephant LH and FSH in yields of 8.8 and 0.48 mg, respectively, per 10 g pituitary powder. The preparations were characterized by HPLC gel filtration and amino acid analysis, both of which showed the elephant LH and FSH to be very similar to ovine LH and FSH. The preparations were also characterized by radioimmunoassays and bioassays for LH and FSH and a radioreceptor assay for FSH. Results showed virtually no cross-contamination of hormonal activities in the elephant LH and FSH preparations. Elephant LH potencies ranged from 50 to 66% of highly purified ovine LH and elephant FSH potencies ranged from 21 to 52% of highly purified ovine FSH in the various assays employed. No evidence was found for any demonstrable intrinsic FSH activity in elephant LH. The assays employed suggest possible usage for making physiological measurements of gonadotropins in the elephant.

Radioiodination of Chicken Luteinizing Hormone without Affecting Receptor Binding Potency1

By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of 1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; 2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and 3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final 125I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. 125I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

Effects of diverse mammalian and nonmammalian gonadotropins in a rat granulosa cell bioassay for follicle-stimulating hormone

The biopotencies of pituitary gonadotropins purified from a marsupial (kangaroo), two avian (ostrich and turkey), a reptile (turtle), an amphibian (bullfrog), and two fish (sturgeon and teleost) species were examined using an in vitro rat granulosa cell bioassay for follicle-stimulating hormone (FSH). Treatment of cultured granulosa cells with increasing concentrations of gonadotropin preparations from these species resulted in dose-dependent increases in estrogen production from negligible amounts to maximal levels of approximately 2–29 ng/culture. The relative biopotencies of these FSH preparations from most potent to least potent were in the order of human > ostrich > Turkey > kangaroo > turtle > sturgeon > bullfrog > teleost with ED 50 values of human 8.7 ng/well; ostrich 10.5 ng/well; turkey 22.5 ng/well; kangaroo 58.2 ng/well; turtle 62.5 ng/well; sturgeon 260 ng/well; bullfrog 750 ng/well; teleots > 1000 ng/well. In contrast, luteinizing hormone (LH) preparations were considerably less effective for ostrich, turkey, kangaroo, turtle, and bullfrog, being six-, five-, three-, and twofold less potent than FSH preparations for the same species, demonstrating the specificity of this assay for FSH. An LH preparation from bullfrog was unable to significantly stimulate estrogen production below 500 ng/ml. Thus, the present in vitro bioassay (GAB) using rat granulosa cells provides a sensitive and specific assay for measuring FSH activities of gonadotropins from diverse mammalian and nonmammalian species.

Effects of Removal of Carboxy-Terminal Extension from Equine Luteinizing Hormone (LH) β-Subunit on LH and Follicle-Stimulating Hormone Receptor-Binding Activities and LH Steroidogenic Activity in Rat Testicular Leydig Cells*

Residues 121-149 of equine LH beta (eLH beta) were removed by a simple mild acid treatment procedure. The modified eLH beta, des(121-149)eLH beta, was isolated by gel permeation chromatography on Sephacryl S-200. Recombination of des(121-149)eLH beta with eLH alpha and ovine LH alpha (oLH alpha) produced LH derivatives as efficiently as recombination with native eLH beta. In rat testicular LH radioligand assay systems employed in this study the potencies of the resulting LH preparations were, in order of decreasing potency: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.82:0.67:0.15:0.02:0.006, eLH tracer; 1:0.88:0.67:0.21:0.02:0.006, hCG tracer). In a horse testicular LH radioligand assay with eLH tracer, only the equine LH derivatives were active, and the order of potencies was the same: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta (1:0.58:0.46). In a rat testicular Leydig cell steroidogenesis assay, eLH was the most active preparation, but the relative potencies of the other preparations remained unchanged: eLH greater than des(121-149)eLH beta:eLH alpha greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.61:0.55:0.27:0.004:0.003). We have previously reported that the hybrid consisting of native eLH beta and oLH alpha was inactive (less than 1%) in LH receptor and steroidogenesis assays. The data reported herein confirm this observation and demonstrate that the absence of LH activity in eLH beta:oLH alpha cannot be attributed to the C-terminal extension on eLH beta, since the des(121-149)eLH beta:oLH alpha hybrid LH is also inactive. Examination of the intrinsic FSH activity of eLH in both rat and chicken testicular FSH radioligand assays produced the following results; eLH, recombined eLH subunits, and des(121-149)eLH beta:eLH alpha were all of the same potency (13% and 0.9% as active as eFSH in rats and chickens, respectively). We conclude that the C-terminal extension on eLH and eCG beta-subunits is not involved in subunit association, LH receptor binding, or FSH receptor binding. The derivative des(121-149)eLH beta:eLH alpha provides a model compound that may be useful in determining the role, if any, of the glycoprotein hormone C-terminal extension that appears to have arisen independently at least twice in mammalian evolution.

The effect of estradiol with Synchro-Mate-B in the superovulation of beef heifers with FSH-P

A study was conducted to determine whether luteal tissue formed after administration of exogenous luteinizing hormone (LH) was capable of maintaining pregnancy in the absence of the original corpus luteum (CL) of pregnancy in heifers. Nineteen beef heifers, confirmed pregnant by ultrasonography approximately 28 days after breeding, were randomly assigned to one of two groups. Nine heifers remained as untreated controls to determine the incidence of spontaneous embryonic or fetal loss. Each of the remaining ten heifers was given two Syncro-Mate-B (SMB) ear implants (12 mg of norgestomet) between 30 and 35 days after breeding (day of SMB implant was Day 1). An additional SMB was implanted on Day 8 and another on Day 15. All implants were removed on Day 20. On Day 2, SMB-implanted heifers were given an intramuscular (i.m.) injection of 500 μg of cloprostenol to induce luteal regression. On Day 5, regression of the CL was confirmed by ultrasonography and these heifers received a highly purified porcine LH preparation (50 mg Armour, i.m.); an additional 25 mg was injected i.m. on Day 6. Ovulation occurred in all ten LH-treated heifers and was detected on Day 7.2±0.1 (mean±SEM). In five of ten LH-treated heifers, the induced CL regressed, and was last detected by ultrasonography on Day 15.8±0.4 (range, Day 15–17). Pregnancy was not maintained in these heifers after SMB implants were removed. Pregnancy was maintained until Day 40 (end of experiment) in the remaining five LH-treated heifers and in all nine control heifers. In all five heifers in which the induced CL regressed, this CL had formed in the ovary contralateral to the original CL of pregnancy. In contrast, in four of five heifers in which the induced CL was maintained, this CL had formed in the ovary ipsilateral to the original CL of pregnancy. Although LH induced ovulation and CL formation in the absence of the CL of pregnancy and in the presence of continuous progestagen support, short-lived CL were formed in five of ten heifers.

Heterogeneity of human luteinizing hormone. Detection and identification of alpha- and beta-subunits in international reference preparations.

The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free alpha- or beta-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free alpha- or beta-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the alpha-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the beta-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM14) which was prepared by one of us and on which we have reported earlier. In addition, with the above-mentioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37 degrees C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, alpha- and beta-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.

Characterization of equine luteinizing hormone by chromatofocusing.

Three equine luteinizing hormone (LH) preparations (eLH-A, -B, and -C) recently have been isolated in our laboratory and were shown to differ in average basicity (eLH-A greater than -B greater than -C). The present study further characterizes these preparations by chromatofocusing. Each of these preparations are comprised of a family of isohormones, with 5 major immunoreactive peaks in the pH range of 7 to 4 (approx. pIs = 6.6, 6.1, 5.7, 5.2, and 4.8), with varying amounts of material eluting to either side of the pH gradient. Although similar isoforms are seen in all three LH preparations, the relative proportions of different isoforms vary in a manner reflecting the average charge properties of eLH-A, -B, and -C. While eLH-A contains predominantly basic forms, eLH-C consists largely of acidic material, and eLH-B is composed mostly of isohormones with pIs intermediate to eLH-A and -C. Chromatofocusing of a crude extract from a single horse pituitary gland revealed isohormone peaks corresponding to those found in the highly purified LH preparations. Peak fractions of the various isoforms were used to generate a variety of activity ratios (LH bioactivity:LH radioimmunoassay (RIA), LH radioreceptorassay (RRA):LH RIA, LH bioactivity:LH RRA, follicle-stimulating hormone (FSH) RRA:LH RIA, and FSH RRA:LH RRA activity ratios). The LH bioactivity:LH receptor binding potency ratio showed a linear increase with increasing isohormone acidity (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation and purification of some glycoproteins by high-performance liqiud chromatography

Native and desialylated human chorionic gonadotrophin showed different elution profiles by size-exclusion high-performance liquid chromatography (HPLC) and the difference was more pronounced using a mobile phase of low ionic strength. Ion-pair reversed-phase HPLC separated components of human chorionic gonadotrophin with apparent molecular weights ranging from 14 000 to 58 000 daltons (using sodium dodecyl sulphate gel electrophoresis). An affinity support prepared by attaching serotonin to LiChrosorb Diol specifically bound components containing sialic acid and may be useful for purification of sialoglycoproteins such as the glycoprotein hormones, β-interferon and transferrin. Size-exclusion HPLC revealed that of two human luteinizing hormone preparations whose sialic acid content correlated with their published in vivo activities, that with higher in vivo activity contained a significant quantity of high-molecular-weight material (possibly aggregation) which was not evident in the sample of lower potency.

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. II. Gel filtration chromatography.

Marked polymorphism was revealed in both stored and circulating forms of immunoreactive follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the bullfrog, Rana catesbeiana, by exclusion chromatography on columns of Sephracyrl-S200. FSH behaved as a more homogeneous and larger molecule than LH from the same pituitary or plasma, but the properties of both hormones in the plasma were markedly affected by gonadectomy. Chromatographic profiles of FSH stored in the pituitaries were similar in intact and gonadectomized frogs, but pituitary LH in the latter was comprised of a larger proportion of early eluting activity. Previously purified preparations of bullfrog FSH and LH were more homogeneous than these extracts. Differences between pituitary hormones in intact and gonadectomized frogs were small compared with those between circulating hormones. Plasma FSH and lH from gonadectomized frogs behaved as more homogeneous and larger molecules than those from intact frogs in which plasma gonadotropins were elevated normally or by injections with gonadotropin releasing hormone (GnRH). Some differences in circulating hormones were also observed between a normal male and female and both differed from gonadectomized an GnRH-treated intact frogs. Chromatographs of plasma gonadotropins in GnRH-treated animals generally resembled those of the hormones stored in the pituitary, whereas plasma FSH and LH in gonadectomized frogs appeared more homogeneous and larger than the pituitary-stored forms. Those pronounced differences in chromatographic properties of gonadotropins in intact and gonadectomized frogs correlate with previously observed effects of gonadectomy on clearance profiles of circulating FSH and LH.

Modification of acute testosterone responsiveness to luteinizing hormone by follicle-stimulating hormone and luteinizing hormone in the domestic cockerel.

Pretreatment of newly hatched cockerels with low doses of highly purified ostrich or ovine follicle-stimulating hormone (FSH) (1 Mg/injection X 5 or 7 injections over 2.5 or 3.5 days) enhanced subsequent acute in vivo testosterone responses to a wide range of doses of NIH ovine luteinizing hormone (LH). Similar pretreatment with corresponding LH preparations or with much higher doses of NIH-LH (25 Mg/injection) enhanced responses to moderate and high doses of NIH-LH, but had no effect on responses to low doses. When combined with FSH in pretreatment, the high doses of NIH-LH suppressed the stimulatory effect of FSH on these latter responses. Other results indicated that the effects of FSH preparations on responsiveness to LH were not due to LH contamination: the activity of the ovine FSH was not reduced by immunoneutralization of its LH contamination, and very low doses of ovine LH, equivalent to amounts that might have contaminated the effective doses of the FSH preparation, had no effect on responsiveness to LH. In slightly older cockerels, pretreatment with highly purified ovine FSH (1 Mg/injection X 14 injection over 7 days, from Days 4-10 after hatching) had effects similar to those in the younger cockerels. Similar pretreatment with NIH-LH (25 Mg/injection) suppressed the acute testosterone response to a low dose of NIH-LH, but seemed to enhance that to a higher dose (though this latter effect was not statistically significant). Thus, in the cockerel, FSH can enhance responsiveness of the testis to acute LH stimulation of testosterone production and LH can also modulate testicular

Development of the rat oocyte in vitro: Inhibition and induction of maturation in the presence or absence of the cumulus oophorus

The effect in vitro of oocyte maturation inhibitors and the ability of preparations of luteinizing hormone to relieve the arrest induced by these substances was studied in preparations of cumulus-free (naked) rat oocytes, and compared to previously obtained results from oocytes enclosed by their cumulus cells. The development of both the cumulus-oocyte complex and naked oocyte is arrested in vitro by cyclic AMP derivatives or cyclic nucleotide phosphodiesterase inhibitors. While gonadotropins can overcome the effect of these substances in the cumulus oocyte complex, they have no effect on naked oocytes. Cholera enterotoxin, an irreversible activator of adenylate cyclase, maintains developmental arrest in cultured cumulus oocyte complexes but not in naked oocytes. Preparations of luteinizing hormone can partially overcome the effect of cholera enterotoxin in the complexes. Furthermore, the acceleration of oocyte maturation in vitro observed in the presence of gonadotropins, which is seen in cumulus oocyte complexes, can be mimicked by stripping the oocyte of its associated cumulus cells. The results of these and other studies suggest that: (1) the cytoplasmic levels of cyclic AMP in the isolated oocyte are high enough to maintain meiotic arrest if a phosphodiesterase inhibitor is present; (2) the oocyte contains an active phosphodiesterase; (3) the oolemma may lack the adenylate cyclase system and; (4) gonadotropins seem to affect the oocyte indirectly, via the cumulus cells, possibly by interrupting communication between the two cell types.

Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey ( Meleagris gallopavo)

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were purified from the pituitary glands of the turkey ( Meleagris gallopavo). These hormones were characterized biochemically and biologically and compared with chicken gonadotropins prepared in several independent laboratories. Amino acid and carbohydrate analyses demonstrated homology between turkey and other species of gonadotropin. The turkey LH purified here had significantly higher carbohydrate content than a previous preparation of turkey LH. Immunological studies further confirmed that the turkey FSH and LH were distinct from one another and that each was homologous to the respective gonadotropin from other vertebrates; the immunopotencies of the turkey hormones were similar to those from chickens. A variety of bioassays and radioreceptor assays (RRAs) confirmed the biological activity of the two turkey gonadotropins and revealed that the turkey LH was distinct from that of the chicken. As expected, the two types of turkey hormones were approximately equipotent in total gonadotropin bioassays (frog spermiation and 32P uptake by chick testes), and only the turkey LH was active in the rat Leydig cell assay and in RRA for LH in mammals. However, the turkey LH was also highly potent in several assays considered to be relatively FSH specific, including the Anolis lizard assay and several RRA systems using mammalian, turtle and avian gonadal receptors with 125I-labeled human FSH as tracer. Turkey and chicken FSH are similar in the RRAs, but the turkey LH was consistently more potent than either avian FSH in competing for FSH-binding sites. Chicken LH had relatively low activity by comparison. It is suggested that the evolution of the structure of active sites in turkey LH has involved convergence on those of the FSH molecule.

Studies on the immunochemical relatedness among tetrapod gonadotropins and their subunits with antisera to sea turtle hormones

Immunological relatedness among gonadotropins from the four classes of tetrapods was studied with three antisera ( a s ) raised in rabbits against follicle-stimulating hormone (FSH), luteinizing hormone (LH), and LHβ subunit from the green sea turtle, Chelonia mydas. Only pituitaries or FSH from turtles showed appreciable cross-reaction in the homologous RIA for Chelonia FSH, and preparations from the heterologous suborder Pleurodira showed weaker and less complete cross-reactivity than from species of Cryptodira. However, the Chelonia FSH a s was capable of neutralizing the biological activity of crocodilian FSH and showed specific binding to 125I-labeled FSH from birds and mammals (but not amphibians). Considerable immunorelatedness was evident among the LH preparations of different species and this appeared to result primarily from common antigenic determinants on the α subunits of LH. First, all chelonian, crocodilian, and avian LH preparations showed high activity in RIA based on anti- Chelonia LH serum and 125I-labeled Chelonia LHα as tracer; in fact, several of these heterologous species of LH were more potent than the Chelonia LH. LH from eutherian mammals (human, ovine, bovine) showed essentially no cross-reactivity, but preparations of ovine LHα and kangaroo LH showed measurable activity in LHα RIA. Second, the antiserum for LH, which contained antibodies to the LHα subunit, neutralized the biological activity of LH from other species of turtles, crocodilians, and a mammal (sheep). Third, 125I-labeled preparations of avian (turkey) and mammalian (ovine and rat) LH showed specific binding to this antiserum. In contrast, parallel tests with an antiserum raised against Chelonia LHβ showed high cross-reactions only with LHs from other species of turtles; i.e., little or no cross-reaction with any nonchelonian species was indicated in RIA, neutralization tests, or binding studies with 125I-labeled hormones. Gonadotropins from amphibians and snakes showed no cross-reaction in any tests with any of the three antisera used. This conspicuous lack of immunological cross-reactivity of snake gonadotropins, especially with antibodies to LHα, provides further evidence for a pronounced structural divergence between the hormones of squamates and the other reptilian orders.