Chemical, biological and immunological properties of pituitary gonadotropins from the donkey (Equus asinus): comparison with the horse (Equus caballus)

Donkey gonadotropins (donkey luteinizing hormone, dLH; donkey follicle-stimulating hormone, dFSH) have been isolated in purified form from 191 donkey pituitaries using essentially the same procedures previously employed for the purification of equine gonadotropins. Chemically, dLH and dFSH were observed to be similar to equine LH (eLH) and FSH (eFSH) in fractionation behavior and glycoprotein nature. Two forms of the dFSH molecule were observed, as is the case for eFSH. Donkey LH had significantly less total carbohydrate (13.5%) and sialic acid (1.9%) than eLH (26.7% and 5.8%, respectively). Carbohydrate (17-21%) and sialic acid (2.4%) content of the two dFSH preparations closely resembled that of eFSH. A slightly higher tyrosine content in the donkey gonadotropins was noted in a comparison of amino acid compositions. Immunologically, in a heterologous FSH radioimmunoassay (RIA), dFSH preparations were equal to or twice as active as eFSH preparations. However, in homologous RIAs for equine chorionic gonadotropin (eCG), eFSH and eLH, both the dLH and dFSH preparations were considerably less active than the equine gonadotropins, and their inhibition curves were all nonparallel. Biologically, in the Steelman-Pohley assay both dFSH preparations were equipotent and as potent as eFSH (approximately 40 times NIH-FSH-S12). In the Sertoli cell assay for cAMP (FSH assay) and the Leydig cell assay for testosterone (LH assay), both dFSH and dLH were 2- or 6-fold more active than eFSH and eLH, respectively. In rat and equine testis FSH homologous radioreceptor assays, dFSH preparations were as active and up to 6-fold more active than eFSH. In contrast, dLH was 10-fold less active than eLH in the equine LH homologous radioreceptor assay. Unlike eLH, dLH was found to possess little intrinsic FSH activity or FSH inhibitory activity, and the small amount of FSH activity observed was most likely due to FSH contamination. Therefore, eLH behaves much like eCG (pregnant mare's serum gonadotropin, PMSG) which also possesses both LH and FSH activity. In contrast, dLH behaves more like donkey chorionic gonadotropin (dCG) which possesses only a low degree of FSH activity.     

Use of the immature rat uterotrophic assay for specific measurements of chorionic gonadotropins and follicle-stimulating hormones in vivo bioactivities

The uterine weight growth stimulation by equine Chorionic Gonadotropin (eCG/PMSG) was found to occur at much lower eCG concentrations than ovarian growth. Human Chorionic Gonadotropin (hCG) which has only LH activity, was found to be as active as eCG in the uterotrophic assay whereas equine Luteinizing Hormone (eLH) which has dual LH+FSH activities like eCG, exhibited a much lower potency. In contrast to hCG, porcine and ovine LH as well as pFSH and oFSH exhibited no uterotrophic activity indicating that only gonadotropins with both LH activity and long half-lives are active alone in this assay. The FSH preparations were nevertheless found to trigger a dose-dependent response, but only in the presence of a subactive dose of hCG. The uterotrophic activity of hCG was found to be suppressed in ovariectomized immature rats and to be diminished after injection of GnRH antagonist suggesting an indirect pathway implicating the hypothalamo-pituitary complex.The data in this report together with the analysis of literature suggest that choriogonadotropins exert their stimulatory role on uterine growth by an indirect mechanism involving an increase in ovarian FSH receptors and FSH release by the pituitary. At the lowest concentrations of hCG, the increase in ovarian FSH receptors without endogenous FSH release is thought to be responsible for the sensitivity of the uterotrophic assay to exogenous FSHs. In conclusion, the immature rat uterotrophic assay is a sensitive and convenient assay for eCG and hCG as well as for FSHs in the presence of a sub-active dose of hCG.     

Equine luteinizing hormone possesses follicle-stimulating hormone activity in hypophysectomized female rats

The ability of equine luteinizing hormone (eLH) to promote follicular growth and maturation in hypophysectomized rats has been assessed. A single injection of equine LH has been shown to promote the growth of a large number of antral and preovulatory follicles. In addition, equine LH markedly increased serum estrogen levels and uterine weight. Furthermore, equine LH, like equine chorionic gonadotropin (eCG; PMSG) was able to significantly enhance the incorporation of [3H]thymidine into ovarian DNA, an activity shown to be specific to hormones having follicle-stimulating hormone (FSH) activity. Equine LH treated with an FSH antibody immunoaffinity column to remove any possible contamination still exhibited the above activity, demonstrating that the FSH activity is intrinsic to the eLH molecule. Equine LH has also been shown to be capable of inducing LH receptors in granulosa cells of ovaries of hypophysectomized rats, an activity specific to FSH-like hormones. From the doses required of eLH and the degree of response observed, it is concluded, however, that eLH in the hypophysectomized rat is less active than eCG as an FSH.     

FSH-induced ovulation in intact and hypophysectomized mice

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

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

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

Zebra chorionic gonadotropin: partial purification and characterization

Six samples of pregnant zebra (z) serum from the first and second trimesters of pregnancy were analyzed by RIA and shown to have chorionic gonadotropin levels comparable to that of the mare (0.9-5.3 micrograms/ml); first trimester levels in most cases were higher than second trimester levels. A pool of the sera (10 ml) was fractionated by methods previously employed for the purification of equine (e) and donkey (d) chorionic gonadotropin to achieve a concentration of the zebra chorionic gonadotropin (zCG). A yield of 1.0 mg of glycoprotein was obtained. HPLC analysis of the material indicated the content of zCG to be about 7%. Its molecular size as judged by Ve/Vo values is smaller than eCG, greater than ovine LH, and about the same as equine LH. The zCG was tested in RIAs for LH and eCG, radioreceptor assays (RRA) for LH and FSH, and the rat testis Leydig cell assay for LH. Comparisons were made with equine and donkey chorionic gonadotropin, and equine and zebra LH. The results, preliminary because the preparation is not of high purity, showed that zCG is bioactive as an LH; immunologically similar to eCG, eLH, dCG, and zLH; and competes in RRAs for LH but not FSH receptors. It differs, therefore, from eCG and eLH--which have high levels of intrinsic FSH activity, and is more like dCG, dLH, and zLH--all of which have minimal if any FSH activity.     

Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin alpha subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin alpha subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F(3) + F(4) granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F(3) + F(4) cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F(1) and F(3) + F(4) granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F(3) + F(4) granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.     

Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. Properties of the Arg beta 43 cleaved hormone

Ovine lutropin (oLH) and its beta subunit (oLH beta) were nicked by short-term incubations with endoproteinase Arg-C. Isolated oLH beta was rapidly nicked and converted from an Mr 18,000 band on sodium dodecyl sulfate-polyacrylamide gels to an Mr 13,000 band. Partial nicking of only the beta subunit in intact oLH was also observed as indicated by the appearance of small amounts of the Mr 13,000 band detected in Arg-C-treated oLH samples. The alpha subunit was protected by association with the beta subunit, but free alpha subunit was rapidly degraded. Sequence analysis of nicked oLH beta indicated that one of the peptide bonds on either side of Arg43 was cleaved by the protease, with a slight preference for the amino side of this residue. Nicked oLH beta was reassociated with oLH alpha, and the resulting dimer was separated from unrecombined subunits. The biologic activity of nicked oLH beta + oLH alpha in an LH radioligand assay was only 2% that of intact oLH.     

Biological and immunoactive substances resembling chorionic gonadotropin are present in full-term horse and zebra placentas.

This study describes the presence of immunoactive and bioactive eCG-like material in full-term placentas of both domestic horses and zebras. Term placental extracts were immunoreactive in an LH monoclonal antibody RIA, and methods successfully used previously for the purification of eCG and eLH were employed to further concentrate the immunoreactive materials to the point where additional characterization studies could be performed. Sufficient equine material was obtained to perform a final fractionation on a concanavalin A Sepharose column yielding an unadsorbed fraction, e17A, and an adsorbed fraction, e17B. There was insufficient zebra material, z5D, for this step. HPLC gel filtration coupled with LH immunoassays of the column eluates showed all the final placental fractions to be highly heterogeneous, but a discrete peak of immunoactivity was found in one of the two equine fractions (e17B) and in the zebra fraction (z5D). The HPLC gel filtration elution volumes for e17B and z5D suggest that they have a smaller molecular size than either eCG or eLH but almost the same size as ovine LH. Both e17B and z5D were bioactive in the rat Leydig cell assay for LH but low in potency compared to eCG or eLH; e17A was inactive at very high doses (5 micrograms). This latter fraction, however, cross-reacted in an eCG alpha RIA to a much greater extent (6 times) than e17B, suggesting that it may be an incompletely formed or degraded alpha subunit. RIAs for LH, eCG, and eCG beta suggest that epitopes distinctive for these molecules are also present or similar to those in the term placental materials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of photoperiod, hypophysectomy, and follicle-stimulating hormone on testicular follicle-stimulating hormone binding sites in golden hamsters

Experiments were conducted to partially characterize and to examine the regulation of unoccupied testicular follicle-stimulating hormone (FSH) binding sites in adult golden hamsters. Testicular FSH binding sites were measured in the 1800 X gav fraction of whole testicular homogenates using iodinated bovine FSH. Binding of FSH was highly specific for FSH, located primarily in the testes, was time- and temperature-dependent, initially reversible, saturable, and consistent with a model consisting of a single class of high-affinity binding sites (range of equilibrium association constants (Ka) 2-12 X 10(10) M-1). Exposure of hamsters to a short photoperiod consisting of 5L:19D was associated with an increase in concentration (fmol/mg protein), but a reduction in total content (fmol/testes) of testicular FSH binding sites. There was no appreciable 5L:19D-associated alteration in receptor affinity (average Ka = 7.83 X 10(10) M-1). Injections of ovine prolactin (oPRL), ovine luteinizing hormone (oLH), or ovine FSH (oFSH) for 3 days into hamsters housed in 5L:19D for 12 wk had no effect on photoperiod-induced changes in testicular FSH binding sites. On Days 5 and 6 post hypophysectomy, a dramatic increase in FSH binding site concentration occurred, with but marginal effects on binding site affinity. Injections of 5 micrograms oFSH on Days 2, 3, and 4 after hypophysectomy prevented the increase in binding site concentrations measured on Day 5. Injection of a combination of 5 micrograms oFSH, 50 micrograms oPRL, and 25 micrograms oLH also reduced testicular FSH binding site concentrations in hypophysectomized hamsters, but oPRL or oLH by themselves were ineffective. The data indicate a homologous down-regulation of testicular FSH binding sites, but do not exclude the involvement of other hormones.     

Deglycosylation of gonadotropins with an endoglycosidase

A commercially available endoglycosidase (N-glycanase, Genzyme, Boston, Mass.) purified from Flavobacterium meningosepticum with a specificity for cleaving asparagine-linked carbohydrate moieties in glycoproteins was tested on several pituitary and chorionic gonadotropins as substrates. All intact hormones tested were resistant to the action of the enzyme as were all beta subunits from the respective gonadotropins. All alpha subunits, however, were susceptible to the enzyme as evidenced by a decrease in molecular size when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparative experiments with ovine luteinizing hormone subunit (oLH alpha) indicated that only 35-40% of the carbohydrate was removed after N-glycanase treatment, suggesting that perhaps only one of the two carbohydrate moieties was cleavable under the conditions employed. The enzyme-modified subunit (DG-oLH alpha) was able to recombine with untreated oLH beta. An in vitro steroidogenic bioassay (rat Leydig cell) showed that the recombinant (DG-oLH alpha-oLH beta) was about 22% as potent as the native oLH, but in a testicular membrane binding assay for LH, it was equal in potency to the native hormone in competing with the radioligand.     

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH)

To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.     

Purification of lutropin and follitropin in high yield from horse pituitary glands

A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitaries, respectively. Subunits were prepared by dissociation in 8 M guanidine HCl followed by either gel filtration (eLH) or gel filtration followed by QAE-Sephadex chromatography (eFSH). The hormones and their subunits were characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, NH2-terminal analysis, and by LH and FSH radioligand receptor assays.     

Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa cells.

Previously described models for avian ovarian steroidogenesis, using mature, 25-40-mm preovulatory follicles as the source of tissues, were based on the assumption that interaction of the granulosa layer, as the predominant source of progesterone, with adjacent theca cells is required for maximal production of C21, C19, and C18 steroids. In the present study, we evaluated the steroidogenic capacity of ovarian cells isolated from less mature, 6-8-mm and 9-12-mm follicles in the chicken ovary (representative of a stage of development 2-3 wk prior to ovulation) to determine at which stage of follicular development granulosa and/or theca cells become steroidogenically competent. Granulosa cells collected from 6-8-mm follicles were found to be virtually incompetent to produce steroids, containing extremely low basal levels of progesterone (12 pg/5 x 10(5) cells) and failing to respond with increased steroid output following a 3-h exposure to ovine LH (oLH; 0.1 and 100 ng/0.5 ml), ovine FSH (oFSH; 100, 500, and 1,000 ng/0.5 ml), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP; 0.33 and 3.33 mM) or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, addition of pregnenolone (20 and 200 ng/0.5 ml) to granulosa incubations resulted in significantly increased progesterone levels. Granulosa cells of 6-8-mm follicles also failed to increase cAMP formation in the presence of oLH (10, 100, and 1,000 ng/0.5 ml) and 3-isobutyl-1-methylxanthine (IBMX; 10 microM), but responded to stimulation with 1,000 ng oFSH (4.4-fold increase over basal) or 10 microM forskolin (32-fold increase over basal) in the presence of IBMX. In contrast, granulosa cells isolated from 9-12-mm follicles and incubated for 3 h in vitro were found to contain basal progesterone levels 200-fold higher than those found in granulosa cells of 6-8-mm follicles. Furthermore, granulosa cells of 9-12-mm follicles markedly increased progesterone production following incubation in the presence of oFSH (100-1,000 ng/0.5 ml), 8-bromo-cAMP (0.33 and 3.33 mM), or 25-hydroxycholesterol (250 and 2,500 ng/0.5 ml). However, these granulosa cells remained unresponsive to oLH (0.1, 10, and 100 ng/0.5 ml), failing to increase cAMP accumulation (in the presence of IBMX) and progesterone output. Theca cells of small yellow follicles were found to produce measurable basal levels of progesterone, androstenedione, and estradiol, and levels of each steroid were significantly increased following a 3-h challenge with oLH, 8-bromo-cAMP, 25-hydroxycholesterol, and pregnenolone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Study of the superactivity of equine follicle-stimulating hormone in in vitro stimulation of rat Sertoli cells

We have previously shown that equine follicle-stimulating hormone (FSH) stimulates plasminogen activator secretion in Sertoli cells at much lower concentrations than would be expected from its relative binding activity. We have introduced the term 'superactivity' to designate this particular behavior. In the present study, we show that equine FSH triggers a long-lasting (20 h) plasminogen activator secretion, whereas rat, porcine and ovine FSH as well as equine LH and equine choriogonadotropin (CG) provoke a short-term response (2.5 h). Moreover, equine FSH was also shown to be superactive in the stimulation of estradiol secretion and cyclic AMP production. This indicates that the step responsible for the long-term stimulation by equine FSH is not located beyond cAMP accumulation. Equine and porcine FSH were found to be equally stable during incubation with the cells demonstrating that equine FSH superactivity was not due to higher stability. Besides, phosphodiesterase inhibition led to a similar increase in the responses to both hormones. This rules out the possibility that equine FSH superactivity is due to less stimulation of phosphodiesterase activity. All these data strongly suggest that equine FSH exhibits superactivity in rat Sertoli cells by stimulating adenylate cyclase activity for a much longer period of time than do all other gonadotropins. The molecular mechanism of this outstanding behavior remains to be elucidated.     

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

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

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.     

Partial purification and characterization of rhinoceros gonadotropins, growth hormone, and prolactin: comparison with the horse and sheep

The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purified fractions of LH, FSH, growth hormone (GH), and prolactin (PRL). LH was readily measured by a rat Leydig cell assay (0.1-1% x equine LH) and an RIA using a monoclonal antibody to bovine LH (6-11% x equine LH). FSH activity detected in the LH by either an FSH RIA or a calf testis radioreceptor assay (RRA) was extremely low. No FSH activity could be detected in the White Rhinoceros pituitary "FSH" fraction, but was readily detected in the Black Rhinoceros fraction (RIA: 0.2% x equine FSH: RRA: 0.8% x equine FSH). The presence of GH and PRL was determined by SDS-PAGE and Western blots. Results showed a single immunoreactive GH band and multiple immunoreactive PRL bands. Adsorption with Concanavalin A-Sepharose indicated that some of the PRL bands are glycosylated.