Progesterone and the distribution of pituitary gonadotropin isoforms in cattle.

The objective of the present study was to determine the relative proportion of gonadotropin isoforms in bovine pituitary glands affected by progesterone. Twelve postpubertal heifers (Swiss-Zebu) were assigned to three groups (n=4): intact animals in the luteal phase of the estrous cycle (diestrus group); ovariectomized heifers with (OVXP) or without progesterone treatment (OVX). Prior to pituitary gland collection, a blood sample was taken from each animal to determine the circulating progesterone concentration. Pituitary protein extractions processed by chromatofocusing were eluted with a pH gradient ranging from 10.5 to 3.5. The LH and FSH eluent was grouped on the basis of the following three criteria: (1) as either a basic (pH>or=7.5), neutral (pH 7.4-6.5) and acid (pHor=10.5-3.5); (3) on the basis of distinct isoforms 12 peaks of which (A-L) were identified for LH and 11 (I-XI) for FSH. The analysis by range of pH and by pH of elution in the OVX and OVXP groups showed no difference in the LH and FSH isoform ratio, but diestrus cattle differs having a greater ratio (p<0.05) of basic LH isoforms (87.5+/-0.4%) and lesser ratio (p<0.05) of acid isoforms (5.4+/-0.7%). In the diestrus group, the ratio of acid FSH isoform increased (62.1+/-1.7%), while neutral isoforms decreased (5.7+/-0.4%, P<0.05). The analysis by isoform type of LH revealed a greater proportion of isoforms C (pH 9.4) and E (pH 9.0) in the groups with circulating progesterone when compared to the OVX group. The heterogeneity of FSH was quantitatively similar in most isoforms in the three groups, with the exception of the predominant isoform (VIII, pH 4.9) that was more abundant in the diestrus group (p<0.05). These results indicate that progesterone with other gonad factors influence the pituitary glicosylation altering the relative proportions of gonadotropin isoforms.

Luteinizing hormone (LH) isoforms in ruminants: characterization and physiological relevance.

The luteinizing hormone (LH) is secreted in multiple molecular forms into the blood stream; however, few studies have characterized the pattern of the circulating LH isoforms in domestic animals during different physiological stages. Most of the publications are related to the pattern of isoforms present in the anterior pituitary gland. This review includes recent evidence concerning the distribution of LH isoforms in the pituitary gland and serum in ruminants. The structural heterogeneity of this hormone is emphasized, including the glycosylation biosynthetic pathway, as well as the different proportions of oligosaccharides that confer particular functional characteristics to the heterodimer. Evidence for a regulating role of GnRH, estradiol and progesterone is discussed.

Caprine luteinizing hormone isoforms during the follicular phase and anestrus.

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 microg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH> or =7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH< or =6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p<0.05) in A (12.0+/-0.8%) as compared with F (5+/-2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH> or =10), which amounted to a percentage of 6.0+/-0.4% of the total observed during A, and 3+/-1% during F (p<0.05). Predominant isoforms in A eluted in the pH range 9.99-9.0 (42+/-3%) as compared to 7+/-3% (p<0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99-8.0, representing 55+/-8%, while in A the proportion was 11+/-2% (p<0.01). Isoforms eluted at the pH range 7.9-7 represented a significantly greater proportion during A (5.0+/-0.6%) as compared with F (3+/-1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.

Ovine serum and pituitary isoforms of luteinising hormone during the luteal phase.

The relative abundance of the different isoforms of pituitary and circulating luteinising hormone (LH) in ewes, at different times after the administration of gonadotrophin-releasing hormone (GnRH), during the luteal phase of the oestrous cycle was investigated. Sixteen ewes on Day 9 of their cycle were divided into four groups (n = 4). The control group (T0) received saline solution; the remaining animals received 100 microg GnRH (i.m.) 30, 90 or 180 min (T30, T90 and T180, respectively) before serum and pituitary gland collection. Luteinising hormone polymorphism was analysed by chromatofocusing (pH 10.5-3.5). The LH eluted from each chromatofocusing was grouped on the basis of the following three criteria: (1) according to the pH of elution (pH > or = 10-3.5); (2) as either a basic (pH > or = 7.5), neutral (pH 7.4-6.5) and acidic (pH < or = 6.4) elution of LH of serum and hypophyseal origin; and (3) on the basis of distinct isoforms, of which 10 (A-J) were identifiable in hypophyseal extracts and four (A-D) were found in the serum. In general, the most abundant forms of LH in both the pituitary and serum, at all times, were basic. However, that proportion was greater in hypophyseal extracts (84 +/- 3%, 81 +/- 4%, 82 +/- 3% and 83 +/- 2% at T0, T30, T90 and T180, respectively) than in serum (51 +/- 5%, 48 +/- 10% and 54 +/- 6% at T30, T90 and T180, respectively). Neutral and acidic LH made up a larger proportion of the total LH in sera (neutral: 17 +/- 4%, 20 +/- 6% and 23 +/- 3% at T30, T90 and T180, respectively; acidic: 32 +/- 8%, 32 +/- 11% and 23 +/- 6% at T30, T90 and T180, respectively) than in the pituitary extracts (neutral: 4.0 +/- 0.7%, 10 +/- 4%, 7 +/- 2% and 5.0 +/- 0.5% at T0, T30, T90 and T180, respectively; acidic: 12 +/- 3%, 11 +/- 2%, 12 +/- 2% and 12 +/- 2% at T0, T30, T90 and T180, respectively) at all times. These data reveal that the relative composition of the LH present in the pituitary gland and the LH secreted into the circulation is different, with more neutral and acidic isoforms being secreted. The pattern of circulating LH isoforms changes between 30 and 180 min after GnRH peak induction, with a greater proportion of isoform C (eluting between pH 7.0 and 6.5) at T180 compared with T30 and T90.

Pattern of circulating luteinizing hormone isoforms during the estrous and luteal phases in Holstein heifers.

The pattern of distribution of circulating luteinizing hormone (LH) isoforms in cattle during estrus and the luteal phase was investigated. In each stage, the stage of the estrous cycle was synchronized in seven Holstein heifers with a prostaglandin analogue. After estrus was detected, blood samples were taken at 2-h intervals for 24h. In the luteal phase, animals received 250 microg i.v. of GnRH and blood samples were collected every 15 min for 5h. LH concentration in the samples was determined. Samples with the greatest LH concentration in estrus (pre-ovulatory peak) and those collected 60 min after GnRH administration (luteal phase) were analyzed by chromatofocusing, eluted with a pH gradient from 10.5 to 3.5. Eluted LH was grouped into basic (pH > or = 7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH < or = 6.4) as well as by pH unit. In both phases, basic forms were the most abundant, and these were greater (P < 0.05) during the luteal phase (78.4 +/- 4.2%) as compared with during estrus (57.1 +/- 6.2%); the proportion of neutral and acidic isoforms in estrus (13.7 +/- 2.6%; 28.5 +/- 2.8%) was greater (P < 0.05) as compared with the luteal phase (3.0 +/- 0.7; 18.7 +/- 3.4). These results indicate that the relative proportion of LH isoforms secreted by the adenohypophysis differ by stage of estrous cycle. The addition of excess of NaCl to the column modifies the antigen-antibody binding in the RIA, and the proteins eluted are erroneously quantified as LH; this is an artifact of the technique.