Biomedical subjects

H E Grotjan

Publications and source records attributed to H E Grotjan.

At least 19 recordsLinked to original sources

PubMed2006

Strategies for construction of luteinizing hormone beta subunit analogs with carboxyl terminal extensions in non-primate, non-equid mammalian species.

Chorionic gonadotropins (CG) are unique because they have a carboxyl terminal peptide (CTP) extension on their beta subunits that prolongs circulatory survival. CGbeta genes from the human being and horse have evolved from ancestral luteinizing hormone (LH) beta genes by different pathways that involve deletions that change the reading frames and yield a CTP. Here we further review our previous analysis, aimed at determining whether LHbeta genes in non-primate, non-equid species inherently possess DNA sequences that encode CTP-like domains. In multiple mammalian species, simple frame-shift mutations using either the human or equine CGbeta gene as a model can be used to construct LHbeta analogs with putative CTP domains. Furthermore, DNA sequences from mammalian LHbeta genes can be aligned to maximize similarity with CGbeta genes in order to devise more refined strategies for construction of CTP-bearing LHbeta analogs as exemplified in the bovine case. Thus, mammalian LHbeta genes have DNA sequences that can be potentially expressed in order to construct CTP-bearing glycoprotein hormone analogs.

Amino Acid Sequence↗

PubMed2006

Homologous and heterologous carboxyl terminal peptide (CTP) linker sequences enhance the secretion of bioactive single-chain bovine LH analogs.

Single chain variants of the heterodimeric gonadotropins were engineered by tethering the genes of the individual subunits into one polypeptide. In tethered human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit serves as an effective linker to enhance the secretion of the analogs compared to variants lacking the CTP. The gonadotropin subunits of non-primate, non-equid species lack a CTP domain that precludes the use of a homologous CTP in tethered analogs in many species. Here we used the bovine LH as a model to examine the impact of the CTP domain of the hCGbeta subunit (denoted as huCTP) and of a previously untranslated CTP-like sequence decoded from the bovine LHbeta gene on the secretion and bioactivity of tethered analogs. This cryptic CTP (designated boCTP) was incorporated into the bovine LHbeta reading frame by deletion frame-shift mutations analogous to these that presumably occurred in primates and equids. We genetically engineered single chain variants in which the beta and alpha subunit domains were linked directly or via the heterologous huCTP or the homologous boCTP sequences and expressed them in CHO cells. The data suggest that the tethered analogs were expressed and N-glycosylated, but unlike the huCTP, the boCTP appears as devoid of mucin O-glycans. The incorporation of the boCTP or huCTP linkers enhanced by about 3fold the rate and efficiency of secretion from the transfected cells. The tether variants were bioactive, as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, the variants were about equally potent, as judged by their EC50s (0.7-0.9 ng/ml). Thus, the hCGbeta CTP maintains pro-secretory determinants without inhibiting receptor activation when applied as a linker in tethered bovine LH, implying that these CTP features are preserved when the domain is incorporated into non-primate single chain analogs. The study suggests that the boCTP and huCTP domains are advantageous for the secretion of tethered bovine gonadotropins, and also demonstrates strategies for the design of bioactive LH analogs in ruminant species.

Amino Acid Sequence↗

PubMed1999

Bioassay for growth hormone releasing hormone (GHRH) using a recombinant receptor and cAMP-responsive reporter system.

Growth hormone releasing hormone (GHRH) receptors are members of the G-protein receptor family that use cAMP as a second messenger. A human fetal kidney 293-derived cell line stably expressing the porcine GHRH receptor (pGHRHr/293 cells) and a cAMP-responsive reporter system were used to develop a bioassay for human GHRH. The reporter system (ph alpha180SEAP) was constructed by subcloning the tandem cAMP response elements from the human glycoprotein hormone alpha subunit gene promoter (h alpha180) upstream from the secreted alkaline phosphatase cDNA of reporter plasmid pSEAP-Basic. To generate a stable cell line expressing both the GHRH receptor and SEAP reporter system, a DNA fragment from pPUR that confers puromycin resistance was subcloned downstream from the reporter construct of ph alpha180SEAP. Tranfection of ph alpha180SEAPpur into pGHRHr/293 cells yielded pGHRHr/SEAP/293 cell lines that responded to recombinant GHRH with dose-dependent increases in SEAP activity. The GHRH receptor-SEAP reporter bioassay was compared to a conventional bioassay using cultured rat anterior pituitary cells. Synthetic and recombinant GHRH induced a 3.1-fold increase in growth hormone release by rat pituitary cells with ED50's of 3.6 and 2.2 x 10(-10) M, respectively. Recombinant GHRH was 1.7 +/- 0.7 times more potent than synthetic GHRH in the pituitary cell bioassay. In an analogous experiment, pGHRHr/SEAP/293 cells responded to synthetic and recombinant GHRH with a 9.1-fold increase in SEAP activity. The ED50's were 7.8 and 4.3 x 10(-11) M, respectively, with recombinant GHRH being 1.8 +/- 0.1 times more potent than the synthetic preparation. Thus, the GHRH receptor-SEAP reporter bioassay is a sensitive, accurate, precise and efficient method for measuring GHRH biological activity.

Animals↗

PubMed1998

Neither castration nor steroid-replacement change the apparent molecular size of FSH in the sheep pituitary.

Gonadal steroids alter the apparent molecular size of intrapituitary Follicle-Stimulating Hormone (FSH) in rats and monkeys as well as increase the percentage of acidic FSH isohormones in sheep. Hence, we hypothesized that the molecular size of ovine (o) FSH would be increased by gonadal steroids. Extracts of pituitaries from rams and wethers, as well as, from wethers which had been implanted with dihydrotestosterone (DHT), 17 beta-estradiol (E2) or both steroids (n = 4-6 per treatment group) were subjected to analytical gel permeation chromatography using Sephadex g-100 Superfine. FSH concentrations in chromatographic fractions were determined by radioimmunoassays. Although FSH in pituitaries of non-implanted wethers eluted slightly earlier (i.e. larger) than FSH in pituitaries from E2-implanted wethers as evaluated by distribution coefficients (Kds) during chromatography (P < 0.05), gonadal steroids did not consistently increase Kds but tended to decrease them. When Kds were extrapolated to apparent molecular weights using a series of standard proteins (bovine serum albumin (bSA), ovalbumin (OA), carbonic anhydrase (CA) and cytochrome c (CC)) that were included in each chromatogram, the differences between treatment groups were not statistically significant (P > 0.05). Thus, in contrast to rats and monkeys, neither castration nor steroid-replacement appears to alter the molecular size of FSH in the sheep pituitary as evaluated by analytical gel permeation chromatography.

Animals↗

PubMed1998

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist.

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.

Animals↗

PubMed1998

Ovine luteinizing hormone heterogeneity: androgens increase the percentage of less basic isohormones.

The heterogeneity of luteinizing (LH) is hormonally regulated with androgens having been reported to increase the percentage of extremely basic LH isohormones in the ovine pituitary. This study re-examined the effects of androgens on the heterogeneity of LH in the ovine pituitary because the extremely basic isoforms of LH reported previously were subsequently demonstrated to be artifacts resulting from the conditions used for chromatofocusing. Pituitary extracts from wethers, rams, and dihydrotestosterone (DHT)-implanted wethers were desalted by flow dialysis and chromatofocused on pH 10.5 to 7.0 gradients. LH was quantitated by radioimmunoassays. When compared with rams, wethers had increased percentages of LH isoforms eluting between pH 9.6 and 10. Patterns of intrapituitary LH isohormones were comparable in rams and DHT-implanted wethers except that rams had a higher percentage of LH eluting at pH 9.2 (20.9 +/- 2.1% vs. 13.3 +/- 1.3%). In contrast to what was previously reported, rams and DHT-implanted wethers had higher percentages of ovine LH isoforms eluting at pH's 9.4 or lower. Thus, androgens alter LH heterogeneity yielding increased percentages of isohormones eluting at pH's of 9.4 or lower.

Androgens↗

PubMed1996

Effects of varying levels of duodenal or midjejunal glucose and 2-deoxyglucose infusion on small intestinal disappearance and net portal glucose flux in steers.

Six crossbred steers (261 +/- 18 kg BW) fitted with hepatic portal, mesenteric venous and arterial catheters, and duodenal, midjejunal, and ileal cannulas were used in a replicated 3 x 3 Latin square design to determine the effect of varying levels and site of glucose plus 2-deoxyglucose (2DG) infusion on net portal-drained visceral flux. Steers were fed chopped alfalfa in six equal portions daily at 1.5% of BW. Glucose (0, 9, or 18 g/h) and 2DG (0, 1, or 2 g/h) were infused continuously through the duodenal or midjejunal cannula (two infusion sites) at total glucose plus 2DG infusion rates of 0, 10, or 20 g/h. Arterial and portal blood samples were taken simultaneously at 20-min intervals from 5 to 9 h of infusion. Portal blood flow was determined by continuous infusion of p-aminohippurate and net flux was calculated as venous-arterial concentration (PA) difference times blood flow. Arterial concentration of glucose was not affected (P > .10) by glucose plus 2DG infusion, whereas arterial concentration of 2DG was greater (P < .05) when glucose plus 2DG was infused into the duodenum and increased (linear, P < .10) as amount of glucose plus 2DG infused into both the duodenum and midjejunum increased. Net portal flux and PA difference of glucose and 2DG were greater (P < .05) when glucose plus 2DG was infused into the duodenum. Although 2DG was infused at 10% of the total glucose plus 2DG infusion, it accounted for only 1.7 and .7% of the glucose plus 2DG appearing in portal blood when glucose plus 2DG was infused at 10 and 20 g/h, respectively. We conclude that glucose is more readily absorbed across the proximal-half than the distal-half of the small intestine, and that passive diffusion is a minor route of glucose absorption.

Animals↗

PubMed1995

Increasing concentrations of 17 beta-estradiol has differential effects on secretion of luteinizing hormone and follicle-stimulating hormone and amounts of mRNA for gonadotropin subunits during the follicular phase of the bovine estrous cycle.

The hypothesis tested was that 17 beta-estradiol (E2) would increase amounts of mRNA for alpha, LH beta, and FSH beta subunits during the follicular phase of the estrous cycle prior to the preovulatory surge of gonadotropins in cows. On Day 16 (Day 0 = estrus) of the estrous cycle, all cows were treated with prostaglandin F2 alpha (PGF2 alpha). Cows served as intact controls (CONT, n = 4) were ovariectomized (OVX, n = 5), or were ovariectomized and administered E2 (OVXE, n = 6) in increasing doses starting at the time of treatment with PGF2 alpha. Cows were bled for 6 h before and for 40 h after PGF2 alpha treatment to characterize pulsatile secretion of LH and FSH. Forty hours after PGF2 alpha treatment, pituitaries were collected for evaluation of amounts of mRNA for alpha, LH beta, and FSH beta subunits. Amplitude of LH pulses was greater (p < 0.05) in cows from the OVXE than from the CONT group. Concentrations of FSH were greater in cows from both the OVXE and OVX (p < 0.01) groups than from the CONT group. Amounts of mRNA for alpha and FSH beta subunits were greater (p < 0.01) in pituitaries of cows from the OVX than from the CONT or OVXE groups. Amounts of mRNA for LH beta subunit in pituitaries of cows from the OVX group tended to be greater (p < 0.08) than from the CONT group. Cows in the OVXE group tended (p < 0.08) to have greater amounts of mRNA for FSH beta subunit than did CONT cows.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effects of 17 beta-estradiol on distribution of pituitary isoforms of luteinizing hormone and follicle-stimulating hormone during the follicular phase of the bovine estrous cycle.

The objective of this study was to examine the influence of 17 beta-estradiol (E2) on distribution of LH and FSH isoforms during the follicular phase of the bovine estrous cycle prior to the preovulatory surges of LH and FSH. On Day 16 of the estrous cycle (Day 0 = estrus), intact controls (CONT; n = 4) were treated with prostaglandin F2 alpha (PGF2 alpha) to induce luteal regression and initiation of the follicular phase. Other cows were also treated with PGF2 alpha and either ovariectomized (OVX; n = 5) or ovariectomized and given E2 implants (OVXE; n = 6) to mimic the pattern of increasing E2 concentrations during the follicular phase of the estrous cycle. Pituitaries were collected 40 h after treatment with PGF 2 alpha or ovariectomy (0 h). Aliquots of pituitary extracts were chromatofocused on pH 10.5-4.0 gradients. The LH resolved into thirteen isoforms (designated A-L and S, beginning with the most basic form) while FSH resolved into nine isoforms (designated I-IX, beginning with the most basic form). The percentage of LH as isoform F (elution pH = 9.32 +/- 0.01) was greater (p < 0.05) in the OVX group (48.5%) than in the OVXE group (45.0%). LH isoforms I (elution pH = 6.98 +/- 0.01) and J (elution pH = 6.48 +/- 0.01) were more abundant (p < 0.05) in cows from the OVXE (2.3 and 5.8%, respectively) than the OVX group (1.4 and 3.7%, respectively). Distribution of LH isoforms in cows from the three groups did not differ (p > 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Differential regulation of gonadotropin synthesis and release in ovariectomized ewes after treatment with a luteinizing hormone-releasing hormone antagonist.

Our working hypothesis was that synthesis and release of LH, but not FSH, were solely dependent on LHRH. Twenty ovariectomized (OVX) ewes were randomly assigned to one of five treatments (n = 4 per group). Ewes were administered a low (10 micrograms/kg) or high (100 micrograms/kg) dose of LHRH antagonist (LHRH-Ant) at 24-h intervals for 3 or 6 days. Control ewes received vehicle (5% mannitol) at 24-h intervals for 6 days. Blood samples were collected every 15 min for 4 h before LHRH-Ant or vehicle and every 2 h during the period of treatment to determine concentrations of LH and FSH. Twenty-four hours after the last treatment with LHRH-Ant or vehicle, anterior pituitaries were collected and divided in half along the midsagittal plane; the number of receptors for LHRH, pituitary content of LH and FSH, and relative amounts of mRNA for alpha, LH beta, and FSH beta subunits were determined. Concentrations of LH in serum decreased (p < 0.05) from 25.4 +/- 4.3 ng/ml before LHRH-Ant to less than 0.5 ng/ml within 4 h after the first treatment of LHRH-Ant and remained low (< 0.5 ng/ml) throughout the study. Serum concentrations of FSH declined gradually during the 3- or 6-day period of treatment with LHRH-Ant, from 37.3 +/- 2.4 and 26.5 +/- 4.8 ng/ml to 19.9 +/- 1.8 and 13.7 +/- 2.1 ng/ml, respectively. The magnitude of decline in serum concentrations of LH and FSH did not differ among ewes treated with low or high doses of LHRH-Ant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Luteinizing hormone has a role in development of fully functional corpora lutea (CL) but is not required to maintain CL function in heifers.

We tested the hypothesis that endogenous pulses of LH have a role in development and maintenance of CL during the estrous cycle of the bovine female. Twenty heifers were synchronized to estrus by treating two times with prostaglandin F2 alpha 11 days apart (Day 0 = behavioral estrus). Heifers were then randomly assigned to one of four treatments (n = 5/group). Heifers were treated with an antagonist to LHRH (LHRH-Ant; N-Ac-D-Nal[2]1,4Cl-D-Phe2,D-Pal[3]3,D-Cit6,D-Ala10- LHR H; 10 micrograms/kg body weight) or vehicle (5% mannitol) once every 24 h: 1) LHRH-Ant Days 2-7, 2) LHRH-Ant Days 7-12, 3) LHRH-Ant Days 12-17, 4) no LHRH-Ant (control). Blood samples were collected from the jugular vein twice daily on Days 0-24, and area under the profile of progesterone in circulation during the luteal phase of the estrous cycle was characterized from the start of each treatment period until the demise of CL or Day 24, whichever came first. Luteolysis was considered to have occurred when three consecutive samples contained less than 1 ng progesterone/ml plasma. Areas under the profile of progesterone in circulation during the luteal phase of the estrous cycle were compared to those of heifers from the control group for the same period. LHRH-Ant treatment diminished LH pulses in all treatment groups compared to control (p < 0.05). Treatment with LHRH-Ant on Days 2-7 diminished function of CL (3.72 +/- 0.93 vs. 7.36 +/- 1.02 units, respectively; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effect of immunization against LHRH on isoforms of LH in the ovine pituitary.

The role of LHRH in modulating intrapituitary LH content as well as the distribution of LH among its isoforms was examined in sheep. Rams (n = 3) and wethers (n = 6) were actively immunized against an LHRH-human serum globulin conjugate. Pituitaries collected from these animals plus pituitaries from corresponding numbers of nonimmunized rams and wethers were extracted with a buffered saline solution containing protease inhibitors. Immunization markedly reduced total amounts of immunoreactive LH in the pituitary. An aliquot of each pituitary extract was desalted by flow dialysis against water and chromatofocused on a pH 10.5-7.0 gradient. Concentrations of LH in chromatofocusing fractions were determined by radioimmunoassay. LH in pituitary extracts resolved into nine peaks during chromatofocusing which were coded with letters beginning with the most basic isoform. The percentage of LH as the two most basic isoforms, A' and B, was similar (P > 0.05) in all treatment groups. Isoform H constituted a higher percentage (P < 0.05) of the LH in both castrate groups. Nonimmunized wethers had higher percentages of isoforms C, D and E (P < 0.05) and lower percentages (P < 0.05) of the acidic isoforms (coded as peak Z herein) than did other treatment groups. Thus, castration shifted the pattern of intrapituitary isohormones towards the more basic forms. Nonimmunized rams had a higher percentage (P < 0.05) of isoform G than did other groups. Isoform F, the most abundant isoform, was present as a higher percentage (P < 0.05) in immunized rams and wethers than in nonimmunized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Steady-state amounts of alpha- and luteinizing hormone (LH) beta-subunit messenger ribonucleic acids are uncoupled from pulsatility of LH secretion during sexual maturation of the heifer.

Our primary objective for this study was to determine whether steady-state amounts of alpha- and LH beta-subunit mRNAs in the anterior pituitary are altered during sexual maturation in the bovine female. A secondary objective was to determine whether 17 beta-estradiol (E2) alters amounts of LH subunit mRNAs before onset of puberty. Heifers (7 mo old) were assigned to one of three treatments: 1) ovariectomized (OVX, n = 16); 2) OVX and administered E2 (OVXE, n = 16); or 3) ovary-intact (INTACT, n = 20). Pituitaries were collected at an estimated 120 days before onset of puberty (prepuberty) or 25 days before onset of puberty (peripuberty). Six INTACT heifers were used to determine time of puberty during the experimental period, and their pituitaries were collected 40 h after administration of prostaglandin F2 alpha (postpubertal INTACT group). Relative amounts of mRNAs for LH subunits in each pituitary were determined by Northern analysis and scanning densitometry. Amounts of alpha- and LH beta-subunit mRNAs were lower in pituitaries of INTACT heifers and OVXE heifers, regardless of stage of sexual maturation, than in those of OVX heifers. Amounts of alpha-subunit mRNA were similar in OVXE and INTACT heifers regardless of stage of sexual maturation. Amounts of LH beta-subunit mRNA did not change during sexual maturation in heifers in the INTACT group. Concentrations of E2 were higher and LH beta-subunit mRNA were lower in heifers from the prepubertal OVXE group than in heifers in all other treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer.

Our working hypotheses for this study were that 1) the profile of intrapituitary LH and FSH isoforms would be shifted toward acidic forms as sexual maturation progresses in the bovine female; and 2) concentration of 17 beta-estradiol (E2) in circulation during sexual maturation would be a major factor modulating the percentage of the more acidic isoforms. In addition, the biological-immunoreactive (B:I) ratios of each isoform of LH were evaluated at selected stages of sexual maturation. Heifers (7 mo of age) were assigned to one of three treatment groups: 1) ovariectomized (OVX; n = 16); 2) OVX and administered E2 (OVXE; n = 16); or 3) ovary-intact (INTACT; n = 14). Pituitaries were collected from heifers in each group at an estimated 120 days (prepubertal) of 25 days before puberty (peripubertal). A fourth group of 6 heifers remained intact (postpubertal INTACT) to determine time of puberty during the experimental period. Pituitaries of heifers assigned to the postpubertal INTACT group were collected during the follicular phase of the first or second estrous cycle postpuberty. Pituitaries were used for determination of relative amounts of gonadotropin isohormones. Tissue extracts of the pituitaries were chromatofocused on pH 10.5-4.0 gradients. The LH of all pituitaries resolved into thirteen isoforms that were designated isoforms A-L, and S, with isoform A the most basic form. Isoforms F and G (basic pH range) were the predominant isoforms of each chromatofocusing profile and comprised 50-60% of the immunoreactive LH. Isoforms J and K were the major isoforms eluting in the acidic pH range.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Bioactive and immunoreactive concentrations of circulating luteinizing hormone during sexual maturation in the bovine.

The characteristics of circulating LH during sexual maturation in cattle were assessed by examining bioactive and immunoreactive LH concentrations, as well as their ratio (B/I ratio). Male and female intact control (CONT), gonadectomized (GNX; at 241 +/- 3 days of age, Day 0 of the study), and gonadectomized animals administered 17 beta-estradiol (GNXE) were evaluated. Serum samples were collected at 15-min intervals for 24 h at 1, 7, 13, 17, 21, 25, 29, 33, 37, and 43 wk subsequent to Day 0. Bioactive LH was assessed with an in vitro bioassay using mouse testicular interstitial cells. In initial experiments, immunoreactive LH was quantified in RIAs using three different antibodies. The two RIAs employing polyclonal antibodies overestimated low LH concentrations, but the absolute values obtained in each of the three assays were highly correlated. Hence, immunoreactive LH was measured in an RIA using monoclonal anti-bovine LH (bLH) (JR-518B7). No significant changes in the B/I ratios were observed during individual pulses of LH secretion. Accordingly, pools consisting of equal volumes of the serial blood samples collected during the 24-h period for each animal at each stage of maturation (pools) were compared. LH B/I ratios for GNX females increased significantly with time (p less than 0.01) and the B/I ratios for GNX males were significantly higher than for GNX females (p less than 0.05). Concentrations of LH in most of the pools for GNXE and CONT animals were extremely low or nondetectable until the later bleeding periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Bovine luteinizing hormone (LH) isoforms and amounts of messenger ribonucleic acid for alpha- and LH beta-subunits in pituitaries of cows immunized against LH-releasing hormone.

Ovariectomized beef cows were actively immunized against LHRH to test the hypothesis that decreased stimulation of gonadotropes would alter the distribution of LH isoforms and amounts of mRNA for subunits of LH in the pituitary. Eight long-term (3 yr) ovariectomized beef cows were randomly assigned to one of two treatments: immunization against LHRH conjugated to human serum globulin (n = 4) and nonimmunization (control, n = 4). Mean concentration of serum LH in cows immunized against LHRH (1.0 +/- .83 ng/ml) was less (p = 0.01) than in control cows (5.0 +/- 0.83 ng/ml). Amounts of alpha- (p = 0.13) and LH beta-subunit (p = 0.10) mRNA tended to be reduced in cows immunized against LHRH compared to control cows. However, weight of the anterior pituitary and concentrations of LH in this gland did not differ (p = 0.90) among cows from the two groups. Pituitary extracts were chromatofocused on pH 10.5-7.0 gradients, and concentrations of LH in eluent fractions were determined by RIA. Extracts of all pituitaries resolved into nine isoforms (designated A through H and Z beginning with the most basic form). Only isoform F (mid-alkaline elution, pH = 8.8) was influenced by treatment (p = 0.05). Cows immunized against LHRH had a greater relative amount of isoform F (42.1 +/- 1.4%) than controls (37.2 +/- 1.4%). In summary, immunization of cows against LHRH altered 1) circulating concentrations of LH, 2) amounts of mRNA for the subunits of LH, and 3) distribution of intrapituitary LH isoforms without changing the concentrations of LH in the anterior pituitary.

Animals↗

PubMed1992

Ovine luteinizing hormone: isoforms in the pituitary during the follicular and luteal phases of the estrous cycle and during anestrus.

Pituitaries were collected from late follicular phase (n = 5), mid-luteal phase (n = 5), and anestrous ewes (n = 4) to assess changes in intrapituitary LH heterogeneity at selected reproductive states. After homogenization, an aliquot of each pituitary extract was desalted by flow dialysis against water and chromtofocused on a pH 10.5 to 4.0 gradient. Concentrations of LH in pituitary extracts and chromatofocusing fractions were determined by RIA. The LH in pituitary extracts resolved into 13 isoforms during chromatofocusing, which were coded with letters beginning with the most basic isoform. Follicular and mid-luteal phase ewes exhibited similar distributions of intrapituitary LH among its isoforms. Relative to follicular and luteal phase ewes, anestrous ewes had lower percentages of isoforms D and E as well as higher percentages of isoforms G, H, J and K. Isoform F, the predominant molecular form of LH, constituted a similar percentage in all treatment groups (P > .05). Thus, the distribution of intrapituitary LH among its isoforms did not change significantly between the mid-luteal and follicular phases of the estrous cycle, but higher percentages of the weakly basic and acidic forms of LH were present during anestrus. These observations suggest that intrapituitary LH heterogeneity changes minimally throughout the estrous cycle of ewes during the breeding season.

Anestrus↗

PubMed1991

Ovine luteinizing hormone. V. Significance of flow-through peaks observed during chromatofocusing as revealed by various methods of sample preparation and application.

In a previous study [Keel et al., Biol, Reprod., 36 (1987) 1102] the ovine luteinizing hormone (oLH) in pituitary extracts was chromatofocused on pH 10.5-7 gradients after equilibration in 25 mM triethylamine-HCl, pH 11.0, by gel permeation. Under these conditions, some immunoreactive oLH flowed through the columns unrestricted and this was interpreted to represent extremely basic isoforms. However, when selected flow-through peaks were re-chromatofocused, each was contaminated with other isoforms of oLH. In order to clarify this dilemma, various methods of sample preparation and application were systematically compared. Consistent with previous observations, variable amounts of the immunoreactive oLH in pituitary extracts equilibrated in triethylamine by gel permeation, dialysis, flow dialysis or ion-retardation chromatography eluted as flow-through peaks when chromatofocused. In contrast, when the ionic components in the pituitary homogenization buffer were removed by these methods as well as ultrafiltration and the proteins were applied to the resin in the elution buffer (1:45 Pharmalyte 8-10.5-HCl, pH 7.0), none of the immunoreactive oLH in pituitary extracts eluted as a flow-through peak. Thus, it appears that oLH eluting as a flow-through peak results from incomplete binding of the hormone to the chromatofocusing resin when applied in triethylamine.

Animals↗