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Caprine luteinizing hormone isoforms during the follicular phase and anestrus
Animal Reproduction Science 100 (2007) 280–290
Caprine luteinizing hormone isoforms during the follicular phase and anestrus S. Rojas-Maya a , E. Gonz´alez-Padilla a , C. Murcia-Mej´ıa a , A. Olivares-Segura b , J. Hern´andez-Cer´on a , G. Perera-Mar´ın a,∗ a
b
Departamento de Reproducci´on, Facultad de Medicina Veterinaria y Zootecnia, UNAM, Cd. Universitaria, CP 04510 M´exico, DF, Mexico Unidad de Investigaci´on en Biolog´ıa del Desarrollo, Hospital de Pediatr´ıa, Centro M´edico Nacional, S. XXI, IMSS, M´exico 06700, DF, Mexico Received 5 April 2006; accepted 31 July 2006 Available online 5 August 2006
Abstract 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 g 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 ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 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 ≥ 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Heterogeneity; Gonadotropin-releasing hormone; Seasonal reproduction; Secretion
∗
Corresponding author. Tel.: +52 56225860. E-mail address: [email protected] (G. Perera-Mar´ın).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.07.010
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1. Introduction The secretion pattern of luteinizing hormone (LH) changes in accordance with the different reproductive stages (Chemineau et al., 1988; Tanaka et al., 1995). During the follicular phase, LH pulse frequency increases in response to increments of circulating estradiol (Caraty et al., 1989; Caraty and Skinner, 1999). In anestrus, LH secretion is reduced as a response to the increment in sensitivity of the hypothalamic–hypophyseal axis to the negative feedback imposed by estradiol (Legan and Karsch, 1979). Luteinizing hormone is a heterogeneous glycoprotein due to the structure of its oligosaccharides (Combarnous, 1988; Baenzinger and Green, 1988; Manzella et al., 1996). This heterogeneity is reflected in its physicochemical properties and its biological and immunological activity (Burgon et al., 1996; Creus et al., 2001; Barrios-De-Tomasi et al., 2002; Mi et al., 2002; Perera et al., 2004). Luteinizing hormone heterogeneity has been studied in ovine (Keel et al., 1987; Zalesky et al., 1992, 1993; Padmanabhan et al., 1992; Hassing et al., 1993; Christianson et al., 1998; Arrieta et al., 2006), bovine (Stumpf et al., 1992; Kojima et al., 1995; Cupp et al., 1995; Perera et al., 2004), caprine (Perera et al., 1996) pituitaries, and recently in bovine (Perera-Mar´ın et al., 2005) and ovine serum (Arrieta et al., 2006). The relative proportion of each isoform group depends on the physiological status (UlloaAguirre et al., 1995, 1999; Cooke et al., 1996, 1997; Padmanabhan et al., 1998). For example, an endocrine environment with high concentrations of circulating estradiol (Stumpf et al., 1992; Kojima et al., 1995; Cooke et al., 1997) is correlated with a higher percentage of acidic LH isoforms and basic FSH isoforms in the pituitary gland and serum. In contrast, the decrease in circulating estradiol concentration (Kojima et al., 1995; Christianson et al., 1998) and increased of the progesterone (Perera-Mar´ın et al., 2005; Arrieta et al., 2006); after gonadectomy (Christianson et al., 1998; Kojima et al., 1995) and an inhibition with GnRH antagonists (Zalesky et al., 1993), favors an increase in the percentage of more basic LH isoforms and more acidic FSH isoforms in pituitary and serum. These findings indicate that gonadal factors and GnRH participate in the regulation of the LH isoforms distribution pattern. Most studies on LH heterogeneity in domestic animals relate to sheep and cattle; in goats, however, the information is limited to the obtention and purification of the two adenohyphophysis proteins with heterogeneous charge, as well as biological and immunological LH activity (Perera et al., 1996), but the regulation of heterogeneity in circulation has not been studied in this specie. The aim of the present study was to examine the proportion relative of circulating LH isoforms in goats, during different stages of the ovarian cycle. 2. Materials and methods 2.1. Experimental protocol and serum collection The study was carried out at an experimental station of the Facultad de Medicina Veterinaria y Zootecnia of the Universidad Nacional Aut´onoma de M´exico, located in the central highland of M´exico, at 1800 m, with a semi-dry temperature climate. Group F: Six cycling crossbred goats (during October, confirmed by serum progesterone concentrations >1 ng/ml and ultrasonography) of age between 2 and 6 years, mean ± S.D. weight 45 ± 5 kg and a body condition score of 3.0 (Delavaud et al., 2000) were used. Estrus was synchronized using two intramuscular injections 0.075 mg prostaglandin (PG) analogue (Preloban, Intervet, M´exico, Santiago Tianguistenco, M´exico; 0.075 mg/ml prostanglandin) 9 days apart.
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Thirty six hours after the second administration of PG, the goats were observed every 4 h to detect estrus, and once detected, blood samples were collected hourly for the next 24 h. Group A: Four goats in seasonal anestrus (during July, confirmed by serum progesterone concentrations <1 ng/ml and ultrasonography), with similar characteristics as far as breed, age, weight and body condition to those used for the follicular phase portion of the study were utilized. Each goat received 100 g of GnRH (Fertagyl, Intervet-M´exico, 0.1 mg/ml) i.v. to increase the circulating LH concentration. Two blood samples were collected with an interval of 1 h between them before GnRH administration; thereafter, samples were collected every 15 min for 5 h. 2.2. Serum samples for chromatofocusing The LH concentrations in sera collected, allowed the individual identification of the preovulatory peak during the follicular phase, and of that induced during anestrus. For the analysis of LH heterogeneity, only the samples with the maximum concentrations in the pre-ovulatory peak or at 150 min after the administration of 100 g i.v. of GnRH per animal were used. The selected samples (4 ml of serum each) were transferred to dialysis membranes (molecular weight cut-off, 12,000–14,000; Spectrum Medical Industries, Los Angeles, CA, USA) and dialyzed at 4 ◦ C for 48 h with successive change of deionized water every 8 h. Dialyzed serum was lyophilized. 2.3. Chromatofocusing Luteinizing hormone isoforms present in serum were separated according to their charge as previously described (Grotjan and Zalesky, 1991; Perera-Mar´ın et al., 2005). Briefly, the selected sample dialyzed and lyophilized (20 mg of protein) was re-suspended in 4 ml Pharmalyte (Pharmacia Biotech, Uppsala, Sweden) (pH 8.0–10.5)–HCl, diluted 1:45 with deionized water, adjusted to pH 7.0 with 5N HCl and applied to a 27 cm × 0.7 cm (i.d.) column pre-packed with an ionicexchange resin (PBE-118), previously balanced with 20 volumes of 0.025 M HCl–triethylamine (pH 11.0) and kept at 4 ◦ C. Eluted fractions (2 ml) were collected with a flow of 7 ml/h. The pH of each fraction was measured, and when pH 7.0 was reached and stable for more than 10 fractions, the elution buffer was changed for polybuffer 74 (Pharmacia, Biotech), diluted 1:8 with deionized water (pH 3.5) with the aim to obtain the proteins eluted at pH 7.0–3.5. Proteins bound to the column after elution at the lower pH (pH 3.5) were recovered by addition of 1.0 M NaCl to the chromatographic column. Fractions eluted with the pharmalyte buffer were neutralized with a 1.1 M Tris–HCl solution, pH 7.4 (200 l), while fractions eluted with polybuffer 74 and NaCl were neutralized with 1.1 M imidazole, pH 7.0 (200 l). All chromatographic techniques were performed with degasified buffers. The LH concentration was determined in each of the fractions subjected to pH neutralization by radioimmunoassay (RIA), as described in the next section. 2.4. Luteinizing hormone radioimmunoassay for serum samples Luteinizing hormone concentration of each of the serum samples collected during the follicular phase and anestrus, as well as in the fractions obtained during chromatofocusing, were quantified in triplicate with a liquid phase RIA system, specific for LH, with 120 h of incubation at 4 ◦ C (Perera-Mar´ın et al., 2005). Briefly: the RIA was developed using NIDDK-oLH-I-2 as tracer, labeled with the IODO-GEN technique (Arrieta et al., 2006). The LH reference preparation corresponded to NIDDK-oLH-I-2 at doses of 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10 ng per tube, and the first antibody (anti-oLH-26) was used at a final dilution of 1:200,000 (after characterization
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and titration). The separation of the bound fraction from the free fraction was performed with a second antibody (donkey anti-rabbit IgG), diluted 1:80, after and incubation at 4 ◦ C for 24 h. The RIA buffer consisted of phosphates (0.05 M, pH 7.2), 0.14 M NaCl and 0.1% (wt./vol.) bovine serum albumin (BSA). The assay sensitivity was 0.03 ng per tube and the intra- and interassay coefficients of variation corresponded to 6.4% and 10.3%, respectively. Evaluation of the specificity of the system has been previously reported (Arrieta et al., 2006). 2.5. Statistics The percentage of LH eluted in each fraction was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic forms (pH ≤ 6.4) as well as into eight ranges of elution pH (pH 3.5–3.99; 4.9–4.99; 5.0–5.99; 6.0–6.99; 7.0–7.99; 8.0–8.99; 9.0–9.99 and ≥10). To normalize the data, the percentage of recovered immunoreactive LH per each group of pH and pH unit were transformed into arc sine (arc sine of the square root of the proportion, corrected to an angle between 0◦ and 90◦ ) and subjected to analysis of variance (ANOVA), considering the effects of the pH group and the physiological status of the goat sampled (follicular phase or anestrus). The statistical analysis was performed with the SAS (Statistical Analysis System) statistics software. 3. Results The LH secretion (mean ± standard error) is depicted in Fig. 1 for the goats during the follicular phase (panel a) and anestrus (panel b). The LH concentration before GnRH administration (group A) was 0.3 ± 0.03 ng/ml, while 30 min after, LH concentration increased to values of 5 ± 2 ng/ml reaching a zenith at 2.5 ± 0.7 h (10 ± 1 ng/ml). The goats in group F showed the pre-ovulatory LH peak at 7 ± 2 h after the detected estrus, with a circulating concentration of 23 ± 10 ng/ml. The elution patterns (mean ± standard error) of sera analyzed with chromatofocusing are depicted in Fig. 2. Analysis by pH unit (Fig. 3) showed a very basic elution isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during the anestrus, and 3 ± 1% during follicular phase (p < 0.05). Predominant isoforms in the anestrus eluted in the pH range 9.99–9.0 and represented a percentage of 42 ± 3%; in the follicular phase it was of 7 ± 3% (p < 0.01). In group F the predominant isoforms eluted in the pH range 8.99–8.0 (55 ± 8%), while in group A the percentage in that pH range was of 11 ± 2% (p < 0.01). Isoforms eluted in the range of pH 7.99–7, were in a significantly more abundant during anestrus (5.0 ± 0.6%) as compared with the follicular phase (3 ± 1%). The percentage of isoforms that eluted between 6.99 and 3.5 of pH were similar in the two physiological conditions (p > 0.05). When isoforms were grouped by basic, neutral and acidic, the analysis (Fig. 4) indicated that there were not significant differences (p > 0.05) in the proportion of basic and acidic isoforms between the anestrous and follicular phases. The basic forms were the most abundant (64 ± 3%; 68 ± 7%), followed by acidic forms (25 ± 2%; 27 ± 7%). The neutral forms, which eluted in more restricted pH range (7.4–6.5), were in the lesser proportion both in the anestrous and follicular phases (12 ± 0.8%; 5 ± 2%, p < 0.05, respectively). 4. Discussion There were no previous studies found on the pattern of heterogeneity of the LH isoforms in the serum of goats during different physiological conditions; the polymorphism of this hormone in that species has only been reported in the pituitary gland (Perera et al., 1996).
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Fig. 1. Distribution profile of goat serum LH obtained during the follicular phase (panel a) and anestrus (panel b). Each graph shows the LH profile mean ± standard error. The samples used for analysis of LH heterogeneity by chromatofocusing corresponded to the zenith of the pre-ovulatory LH peak or at 150 min after the administration of 100 g i.v. of GnRH per animal. The samples obtained from each animal were collected every 1 h after detection of estrous through 24 h post-estrous, and at 15 min intervals for 5 h after administration of GnRH in the anestrous.
The present study analyzed the serum LH isoforms distribution in two physiological conditions (anestrus and follicular phase) in the goat, characterized by different regulation mechanisms and patterns of secretion (Caraty et al., 1989; Karsch et al., 1993; Caraty and Skinner, 1999). In both physiological conditions, the qualitative chromatofocusing elution pattern of circulating LH isoforms was similar, which coincides with previous observations in the bovine (Perera-Mar´ın et al., 2005) and ovine (Arrieta et al., 2006) species sera. In this study, the total amount of LH in each sample was considered only that contained in the fractions that eluted before the ionic force (1 M NaCl) was applied to the chromatographic column, which differs with other studies that also considered as LH that measured after NaCl (Castro-Fern´andez et al., 2000; Padmanabhan et al., 1992; Hassing et al., 1993). The reason for this decision was based on the demonstration that the protein eluting after 1 M NaCl passes trough
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Fig. 2. Distribution pattern of LH during chromatofocusing separation of sera obtained during the follicular phase (panel a) or anestrus (panel b) of goats. Detailed patterns of LH distribution mean ± standard error are depicted. Twenty milligrams of protein were applied to the ionic exchange column (PBE-118), balanced with 0.025 M triethylamine, pH 11.0, and the column was eluted with a pH gradient of 10–3.5 and 160 (2 ml) fractions were collected. The arrows indicate the time at which the buffer changed during the chromatographic evaluation and after application of 1 M NaCl.
the column, is collected in a solution that alters the antigen–antibody reaction, leaving a great amount of labeled hormone as free, which is erroneously interpreted as a displacement by the endogenous hormone (Perera-Mar´ın et al., 2005). Another methodological aspect that deserves some discussion is that related to the manner the physiological conditions studied were sampled: the anestrus is characterized by LH pulses of low frequency and high amplitude, and lesser mean serum concentrations of LH owing to the negative feedback imposed by estradiol on the secretion of this hormone (Caraty et al., 1989; Karsch et al., 1993). An examination of the relative abundance of different circulating LH isoforms during anestrus would have not been feasible due to the sensibility of the current methodology. Therefore, a single dose of GnRH was administered to provoke a peak of circulating LH during anestrus to be able to have enough LH in the serum to divide it into 160 fractions with measurable quantities in each of the fractions.
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Fig. 3. Percentage of distribution of circulating LH isoforms in goats by unit pH. Results show the percentage ± standard error of each group of isoforms. Serum samples during the estrous and anestrus were analyzed by chromatofocusing, eluted at pH range of 10.5–3.5. For the anestrus, samples were those collected 150 min after 100 g of i.v. GnRH, while the samples collected during estrous were those collected during the zenith of the pre-ovulatory LH peak. The notations a and b denotes differences (p < 0.05) between phases.
It may be argued that the administration of a single i.v. dose of GnRH may modify the characteristics of the LH commonly secreted during anestrus, which cannot be discarded if GnRH induces de novo synthesis in the gonadotroph in a very short time. However, although it has been documented that pulsatile infusion of GnRH analogues increases the proportion of basic serum LH isoforms (Wide and Bakos, 1993) and the transcription of the gene for LH (Burger et al., 2002), studies using a single dose of GnRH in vitro (Zaleky and Grotjan, 1991) and in vivo (Zambrano et al., 1995; Castro-Fern´andez et al., 2000; Perera-Mar´ın et al., 2005; Arrieta et al., 2006) found that there were no changes in the pattern of secreted isoforms of LH. In ovine
Fig. 4. Percentage of distribution of circulating LH isoforms in goats by group pH. Results show the percentage ± standard error of each group of isoforms. Serum samples during the estrous and anestrus were analyzed by chromatofocusing, eluted at pH range of 10.5–3.5. For the anestrus, samples were those collected 15 min after 100 g of i.v. GnRH, while the samples collected during estrus were those collected during the zenith of the pre-ovulatory LH peak. The notation a and b denotes differences (p < 0.05) between phases.
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pituitaries, Brown and McNeilly (1999) determined that, under the stimulus of a single dose of GnRH in the luteal phase, the mRNA for the -subunit of LH was unstable. Additional evidence revealed that the administration of an i.v. bolus of GnRH at diestrus did not increase the mRNA for the GnRH receptor in the gonadotroph (Cowley et al., 1998). This indicates that the acute effect of the GnRH hardly involves de novo LH synthesis, suggesting that, at this stage, GnRH only participates in the release of LH already present in the secretory granules of the gonadotrophs. Therefore, the results of the present study allow us to infer that serum LH analysed during the anestrus of the caprine species, under the experimental conditions imposed, basically correspond to the preformed hormone with the features of the hormone physiologically secreted at this stage. The presence of greater relative amounts of more basic isoforms during anestrous condition as compared with the pre-ovulatory peak, is in agreement with other reports where under low estradiol concentrations, there is a larger proportion of basic LH isoforms. This has been observed in both serum (Perera-Mar´ın et al., 2005; Arrieta et al., 2006) and the pituitary gland (PereraMar´ın, 2003; Arrieta et al., 2006) during the bovine and ovine luteal phases; in pituitary extracts of gonadectomized rams (Christianson et al., 1998) and in ovine and bovine females immunized with antibodies against GnRH (Stumpf et al., 1992; Zalesky et al., 1993). This may indicate that the percentage of basic isoforms in the pituitary and serum increases when the estradiol/progesterone ratios are small (Ulloa-Aguirre et al., 1992) or there is a decreased secretion of GnRH. In contrast, in the follicular phase, including the pre-ovulatory peak, the distribution of LH isoforms tended to be less basic. The physiological importance of the relative abundance of the different isoforms of LH is related to their differences in biological activity. Norris et al. (1989), found that the in vitro biological activity of serum LH in estrous ewes was less when compared with LH during the other phases of the estrous cycle. On the other hand, the in vivo activity is higher for the more acidic isoforms (Fiete et al., 1991) because their clearance rate from circulation is slower (Drickamer, 1991; Baezinger et al., 1992) and because there are differences in the interaction with the specific receptors, depending on the structure of the carbohydrates of the LH molecule. Estradiol participation in gonadotropin glycosylation has been documented. For example, the estradiol modifies in various ways the regulation of gonadotropins’ synthesis and secretion (Ulloa-Aguirre et al., 1988, 1995; Robertson et al., 1991; Ropelato et al., 1999): influencing GnRH synthesis and frequency of secretion (Gharib et al., 1990; Cowley et al., 1998; Nett et al., 2002); inducing the synthesis and quantity of mRNA that codes for the ␣ and LH subunits (Gharib et al., 1990; Hamernik et al., 1995; Di Gregorio and Nett, 1995); regulating glycosylation in the follicular phase, to decrease the activity of N-acetylgalactosamine transferase and GalNac-4-Osulfotransferase, responsible for the incorporation of galactose and sulfate into the gonadotropin N-linked oligosaccharides (Dharmesh and Baenzinger, 1993; Helton and Magner, 1994; Dami´anMatsumura et al., 1999). On the contrary, greater blood concentrations of progesterone, as those observed during the luteal phase, have an opposite effect that can lead to an increase in the presence, and activity, of enzymes responsible for the incorporation of N-acetylgalactosamine and sulfates to the oligosaccharides residues of LH. This communication contributes evidence of the relative abundance of different LH circulating isoforms during the estrous and anestrous phases in the goat. The LH isoforms tend to be more basic during anestrus and this may be related to the effect of estradiol. Acknowledgements The authors wish to thank the National Council for Science and Technology (CoNaCyT) for financing the project (25748-B). We are also grateful to the National Institute of Health (NIH) for
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the donation of hormones for the characterization of isoforms and to the Neurobiology Institute UNAM for the donation of the first antibody used in this study. Susana Rojas Maya is a graduate student at UNAM. Isabel P´erez Montfort translated the manuscript to English. References Arrieta, E., Porras, A., Gonz´alez-Padilla, E., Murcia, C., Rojas, S., Perera-Mar´ın, G., 2006. Ovine serum and pituitary isoforms of luteinising hormone during the luteal phase. Reprod. Fert. Develop. 18, 485–495. Baenzinger, J.U., Green, E.D., 1988. Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim. Biophys. Acta 947, 287–306. Baezinger, U.J., Kumar, S., Brodbeck, R.M., Smith, P.L., Beranek, M.C., 1992. Circulatory half-life but not interaction with the lutropin/chorionic gonadotropins receptor is modulated by sulfation of bovine lutropin oligosaccharides. Proc. Natl. Acad. Sci. USA 89, 334–338. Barrios-De-Tomasi, J., Timosi, C., Merchant, H., Quintanar, A., Av´alos, J.M., Andersen, C.Y., Ulloa-Aguirre, A., 2002. Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones. Mol. Cell. Endocrinol. 186, 189–198. Brown, P., McNeilly, A.S., 1999. Transcriptional regulation of gonadotropin genes. Rev. Reprod. 4, 117–124. Burger, L.L., Dalkin, A.C., Aylor, K.W., Haisenleder, D.J., Marshall, J.C., 2002. GnRH pulse frequency modulation of gonadotropin subunit gene transcription in normal gonadotropes-assessment by primary transcript assay provides evidence for roles of GnRH and follistatin. Endocrinology 143, 3243–3249. Burgon, P.G., Stanton, P.G., Robertson, D.M., 1996. In vivo bioactivities and clearance patterns of highly purified human luteinizing hormone isoforms. Endocrinology 137, 4827–4836. Caraty, A., Locatelli, A., Martin, G.B., 1989. Biphasic response in the secretion gonadotrophin-releasing hormone in ovariectomized ewes injected with estradiol. J. Endocrinol. 123, 375–382. Caraty, A., Skinner, D.C., 1999. Progesterone priming is essential for the full expression of the positive feedback effect of estradiol in inducing the preovulatory gonadotropin-releasing hormone surge in the ewe. Endocrinology 140, 165–170. Castro-Fern´andez, C., Olivares, A., Soderlund, D., L´opez-Alvarenga, J.C., Zambrano, E., Veldhuis, J.D., Ulloa-Aguirre, A., M´endez, J.P., 2000. A preponderance of circulating basic isoforms is associated with decreased plasma half-life and biological to immunological ratio of gonadotropin-releasing hormone-releasable luteinizing hormone in obese men. J. Clin. Endocrinol. Metab. 85, 4603–4610. Chemineau, P., Martin, G.B., Saumande, J., Normant, E., 1988. Seasonal and hormonal control of pulsatile LH secretion in the dairy goat (Capra hircus). J. Reprod. Fert. 83, 91–98. Christianson, S.L., Zalesky, D.D., Grotjan, H.E., 1998. Ovine luteinizing hormone heterogeneity: androgens increase the percentage of less basic isohormone. Domest. Anim. Endocrinol. 15, 87–92. Combarnous, Y., 1988. Stucture and structure–function relationships in gonadotropins. Reprod. Nutr. Develop. 28, 211–228. Cooke, D.J., Crowe, M.A., Roche, J.F., Headon, D.R., 1996. Gonadotrophin heterogeneity and its role in farm animal reproduction. Anim. Reprod. Sci. 41, 77–99. Cooke, D.J., Cowley, M.A., Roche, J.F., 1997. Circulating FSH isoforms patterns during recurrent increases in FSH throughout the oestrus cycle of heifers. J. Reprod. 110, 339–345. Cowley, M.A., Rao, A., Wright, P.J., Illing, N., Millar, R.P., Clarke, I.J., 1998. Evidence for differential regulation of multiple transcripts of the gonadotropin releasing hormone receptor in the ovine pituitary gland; effect of estrogen. Mol. Cell. Endocrinol. 146, 141–149. Creus, S., Chaia, Z., Pellizzari, E.H., Cigorraga, S.B., Ulloa-Aguirre, A., Campo, S., 2001. Human FSH isoforms: carbohydrate complexity as determinant of in-vitro bioactivity. Mol. Cell. Endocrinol. 174, 41–49. Cupp, A.S., Kojima, F.N., Roberson, M.S., Stumpf, T.T., Wolfe, M.W., Werth, L.A., Kittok, R.J., Grotjan, H.E., Kinder, J.E., 1995. Increasing concentration of 17-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. Biol. Reprod. 52, 288–296. Dami´an-Matsumura, P., Zaga, V., Maldonado, A., S´anchez-Hern´andez, C., Timossi, C., Ulloa-Aguirre, A., 1999. Oestrogens regulate pituitary ␣2,3-sialyltransferase messenger ribonucleic acid levels in the female rat. J. Mol. Endocrinol. 23, 153–165. Delavaud, C., Bocquier, F., Chilliard, Y., Keisler, D.H., Gertler, A., Kann, G., 2000. Plasma leptin determination in ruminants: effect of nutritional status and body fatness on plasma leptin concentration assessed by a specific RIA in sheep. J. Endocrinol. 165, 519–526.
S. Rojas-Maya et al. / Animal Reproduction Science 100 (2007) 280–290
289
Dharmesh, S.M., Baenzinger, J.U., 1993. Estrogen modulates expression of the glycosyltransferases that synthesize sulfated oligosaccharides of lutropin. Proc. Natl. Acad. Sci. USA 90, 11127–11131. Drickamer, K., 1991. Clearing up glycoprotein hormone. Cell 67, 1029–1032. Di Gregorio, G.B., Nett, T., 1995. Estradiol and progesterone influence the synthesis of gonadotropins in the absence of gonadotropin-releasing hormone in the ewe. Biol. Reprod. 53, 166–172. Fiete, D., Srivastava, V., Hindsgaul, O., Baenzinger, J.U., 1991. A hepatic reticuloendothelial cell receptor specific for SO4 -4GalNAc beta1, 4GlcNAc beta1, 2Man alpha that mediates rapid clearance of lutropin. Cell 67, 1103–1110. Gharib, S.D., Wierman, M.E., Shupnik, M.A., Chin, W.W., 1990. Molecular biology of the pituitary gonadotropins. Endocr. Rev. 11, 177–198. Grotjan, H.E., Zalesky, D.D., 1991. Ovine luteinizing hormone VI. Analysis of the misclassification errors in the separation of intrapituitary isohormones by chromatofocusing. J. Chromatogr. A 549, 153–158. Hamernik, D.L., Clay, C.M., Turzillo, A., Van Kirk, E.A., Moss, G.E., 1995. Estradiol increases amounts of messenger ribonucleic acid for gonadotropin-releasing hormone receptors in sheep. Biol. Reprod. 53, 179–185. Hassing, J.M., Kletter, G.B., I’Anson, H., Wood, R.I., Beitins, I.Z., Foster, D.L., Padmanabhan, V., 1993. Pulsatile administration of gonadotropin-releasing hormone does not alter the follicle-stimulating hormone (FSH) isoforms distribution pattern of pituitary or circulating FSH in nutritionally growth-restricted ovariectomized lambs. Endocrinology 132, 1527–1536. Helton, T.E., Magner, J.A., 1994. Sialytransferase messenger ribonucleic acid increases in thyrotrophs of hypothyroid mice: an in situ hibridization study. Endocrinology 134, 2347–2352. Karsch, F.J., Dahl, G.E., Evans, N.P., Manning, J.M., Mayfiel, K.P., Moenter, S.M., Foster, D.L., 1993. Seasonal changes in gonadotropin-releasing hormone secretion in the ewe-alteration in response to the negative feedback action of estradiol. Biol. Reprod. 49, 1377–1383. Keel, B.A., Schanbacher, B.D., Grotjan Jr., H.E., 1987. Ovine luteinizing hormone. I. Effect of castration and steroid administration on the charge heterogeneity of pituitary luteinizing hormone. Biol. Reprod. 36, 1102–1113. Kojima, F.N., Cupp, A.S., Stumpf, T.T., Zalesky, D.D., Roberson, M.S., Werth, L.A., Wolfe, M.W., Kittok, R.J., Grotjan, H.E., Kinder, J.E., 1995. Effects of 17 -estradiol on distribution of pituitary isoforms of luteinizing hormone and follicle-stimulating hormone during the follicular phase of the bovine estrous cycle. Biol. Reprod. 52, 297–304. Legan, J.S., Karsch, J.F., 1979. Neuroendocrine regulation of the estrous cycle and seasonal breeding in the ewe. Biol. Reprod. 20, 74–85. Manzella, S.M., Hooper, L.V., Baenzinger, J.U., 1996. Oligosaccharides containing 1,4-linked N-acetylgalactosamine, a paradigm for protein-specific glycosylation. J. Biol. Biochem. 271, 12117–12120. Mi, Y., Shapiro, S.D., Baenzinger, J.U., 2002. Regulation of lutropin circulatory half-life by the mannose/Nacetylgalactosamine-4-SO4 receptor is critical for implantation in vivo. J. Clin. Invest. 109, 269–276. Nett, T.M., Turzillo, A.M., Baratta, M., Rispoli, L.A., 2002. Pituitary effects of steroid hormones on secretion of folliclestimulating hormone and luteinizing hormone. Domest. Anim. Endocrinol. 23, 33–42. Norris, T.A., Weesner, G.D., Shelton, J.M., Harms, P.G., Forrest, D.W., 1989. Biological activity of LH during the peripartum period and the estrous cycle of the ewe. Domest. Anim. Endocrinol. 6, 25–33. Padmanabhan, V., Mieher, C.D., Borondy, M., I‘Anson, H., Wood, R.I., Landefeld, T.D., Foster, D.L., Beitins, I.Z., 1992. Circulating bioactive follicle-stimulating hormone and less acidic follicle-stimulating hormone isoform during experimental induction of puberty in the female lamb. Endocrinology 131, 213–220. Padmanabhan, V., Lee, J.S., Beitins, I.Z., 1998. Follicle-stimulating isohormones: regulation and biological significance. In: Thacher, W.W., Inskeep, E.K., Niswender, R.D., Doborska. C. (Eds.), Reproduction in domestic ruminants. J. Reprod. Fert. 54, 87–99. Perera, M.G., Ortiz, R.F., Gamboa, V.J.J., Reynoso, M.W., Falc´on, A.A., Salas, V.A., 1996. Obtenci´on, purificaci´on y caracterizaci´on de dos formas de hormona luteinizante de la adenohip´ofisis caprina (gLH). Vet. Mex. 27, 1–10. Perera-Mar´ın, G., 2003. Patr´on de distribuci´on de isoformas de la hormona luteinizante (LH) intrahipofisaria y serica en la especie bovina. PhD. Thesis, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´onoma de M´exico, M´exico D.F. Perera, M.G., Falc´on, A.A., Murcia, M.C., Hern´andez, C.J., Gonz´alez, P.E., 2004. Purification of five bovine luteinizing hormone (bLH) isoforms. Chemical, physical, biological and immunological characterization. Vet. Mex. 35, 129–145. Perera-Mar´ın, G., Murcia, C., Rojas, S., Hern´andez-Cer´on, J., Gonz´alez-Padilla, E., 2005. Pattern of circulating luteinizing hormone isoforms during the estrous and luteal phases in Holstein heifers. Anim. Reprod. Sci. 86, 53–69. Robertson, D.M., Foulds, L.M., Fry, R.C., Cummins, J.T., Clarke, I., 1991. Circulating half-lives of follicle-stimulating hormone and luteinizing hormone in pituitary extracts and isoforms fractions of ovariectomized and intact ewes. Endocrinology 129, 1805–1813.
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Ropelato, M.G., Garc´ıa-Rudaz, M.C., Castro-Fern´andez, C., Ulloa-Aguirre, A., Escobar, M.E., Barontini, M., Veldhuis, J.D., 1999. A Prepronderance of basic luteinizing hormone (LH) Isoforms accompanies inappropriate hypersecretion of both basal and pulsatile LH in adolescents with polycystic ovarian s´ındrome. J. Clin. Endorcinol. Metab. 84, 4629–4636. Stumpf, T.T., Roberson, M.S., Wolfe, M.W., Zalesky, D.D., Cupp, A.S., Werth, L.A., Kojima, N., HejL, K., Kittok, R.J., Grotjan, H.E., Kinder, J.E., 1992. A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer. Biol. Reprod. 46, 442–450. Tanaka, T., Takanori, O., Hopshino, K., Mori, Y., 1995. Changes in the gonadotropin-releasing hormone pulse generator activity during the estrous cycle in the goat. Neuroendocrinology 62, 553–561. Ulloa-Aguirre, A., Espinoza, R., Dami´an-Matsumura, P., Larrea, F., Flores, A., Morales, I., D´ominguez, R., 1988. Studies on the microheterogeneity of anterior pituitary follicle-stimulating hormone in the female rat. Isoelectric pattern throughout the estrous cycle. Biol. Reprod. 38, 70–78. Ulloa-Aguirre, A., Cravioto, A., Damian-Matsumura, P., Jimenez, M., Zambrano, E., D´ıaz-S´anchez, V., 1992. Biological characterization of the natural ocurring analogoues of intrapituitary human follicle-stimulating hormone. Hum. Reprod. 7, 23–30. Ulloa-Aguirre, A., Midgley, A.R., Beitins, I.Z., Padmanabhan, V., 1995. Follicle-stimulating isohormones: characterization and physiological relevance. Endocr. Rev. 16, 765–787. Ulloa-Aguirre, A., Timossi, C., Dami´an-Matsumura, P., Dias, J.A., 1999. Role of glycosylation in function of folliclestimulating hormone. Endocrine 11, 205–215. Wide, L., Bakos, O., 1993. More basic forms of both human follicle-stimulating hormone and luteinizing hormone in serum at midcycle compared with the follicular or luteal phase. J. Clin. Endocrinol. Metab. 76, 885–889. Zaleky, D.D., Grotjan, H.E., 1991. Comparison of intracellular and secreted isoforms of bovine and ovine luteinizing hormone. Biol. Reprod. 4, 1016–1024. Zalesky, D.D., Nett, T.M., Grotjan, H.E., 1992. Ovine luteinizing hormone: isoforms in the pituitary during the follicular and luteal phases of the estrous cycle and during anestrous. J. Anim. Sci. 70, 3851–3856. Zalesky, D.D., Schanbacher, B.D., Grotjan, H.E., 1993. Effect of immunization against LHRH on isoforms of LH in the ovine pituitary. J. Reprod. Fert. 99, 231–235. Zambrano, E., Olivares, A., M´endez, J.P., Guerrero, L., D´ıaz-Cueto, L., Veldhuis, J.D., Ulloa-Aguirre, A., 1995. Dynamics of basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoforms distribution throughout the human menstrual cycle. J. Clin. Endocrinol. Metab. 80, 1647–1656.
Caprine luteinizing hormone isoforms during the follicular phase and anestrus S. Rojas-Maya a , E. Gonz´alez-Padilla a , C. Murcia-Mej´ıa a , A. Olivares-Segura b , J. Hern´andez-Cer´on a , G. Perera-Mar´ın a,∗ a
b
Departamento de Reproducci´on, Facultad de Medicina Veterinaria y Zootecnia, UNAM, Cd. Universitaria, CP 04510 M´exico, DF, Mexico Unidad de Investigaci´on en Biolog´ıa del Desarrollo, Hospital de Pediatr´ıa, Centro M´edico Nacional, S. XXI, IMSS, M´exico 06700, DF, Mexico Received 5 April 2006; accepted 31 July 2006 Available online 5 August 2006
Abstract 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 g 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 ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 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 ≥ 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Heterogeneity; Gonadotropin-releasing hormone; Seasonal reproduction; Secretion
∗
Corresponding author. Tel.: +52 56225860. E-mail address: [email protected] (G. Perera-Mar´ın).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.07.010
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1. Introduction The secretion pattern of luteinizing hormone (LH) changes in accordance with the different reproductive stages (Chemineau et al., 1988; Tanaka et al., 1995). During the follicular phase, LH pulse frequency increases in response to increments of circulating estradiol (Caraty et al., 1989; Caraty and Skinner, 1999). In anestrus, LH secretion is reduced as a response to the increment in sensitivity of the hypothalamic–hypophyseal axis to the negative feedback imposed by estradiol (Legan and Karsch, 1979). Luteinizing hormone is a heterogeneous glycoprotein due to the structure of its oligosaccharides (Combarnous, 1988; Baenzinger and Green, 1988; Manzella et al., 1996). This heterogeneity is reflected in its physicochemical properties and its biological and immunological activity (Burgon et al., 1996; Creus et al., 2001; Barrios-De-Tomasi et al., 2002; Mi et al., 2002; Perera et al., 2004). Luteinizing hormone heterogeneity has been studied in ovine (Keel et al., 1987; Zalesky et al., 1992, 1993; Padmanabhan et al., 1992; Hassing et al., 1993; Christianson et al., 1998; Arrieta et al., 2006), bovine (Stumpf et al., 1992; Kojima et al., 1995; Cupp et al., 1995; Perera et al., 2004), caprine (Perera et al., 1996) pituitaries, and recently in bovine (Perera-Mar´ın et al., 2005) and ovine serum (Arrieta et al., 2006). The relative proportion of each isoform group depends on the physiological status (UlloaAguirre et al., 1995, 1999; Cooke et al., 1996, 1997; Padmanabhan et al., 1998). For example, an endocrine environment with high concentrations of circulating estradiol (Stumpf et al., 1992; Kojima et al., 1995; Cooke et al., 1997) is correlated with a higher percentage of acidic LH isoforms and basic FSH isoforms in the pituitary gland and serum. In contrast, the decrease in circulating estradiol concentration (Kojima et al., 1995; Christianson et al., 1998) and increased of the progesterone (Perera-Mar´ın et al., 2005; Arrieta et al., 2006); after gonadectomy (Christianson et al., 1998; Kojima et al., 1995) and an inhibition with GnRH antagonists (Zalesky et al., 1993), favors an increase in the percentage of more basic LH isoforms and more acidic FSH isoforms in pituitary and serum. These findings indicate that gonadal factors and GnRH participate in the regulation of the LH isoforms distribution pattern. Most studies on LH heterogeneity in domestic animals relate to sheep and cattle; in goats, however, the information is limited to the obtention and purification of the two adenohyphophysis proteins with heterogeneous charge, as well as biological and immunological LH activity (Perera et al., 1996), but the regulation of heterogeneity in circulation has not been studied in this specie. The aim of the present study was to examine the proportion relative of circulating LH isoforms in goats, during different stages of the ovarian cycle. 2. Materials and methods 2.1. Experimental protocol and serum collection The study was carried out at an experimental station of the Facultad de Medicina Veterinaria y Zootecnia of the Universidad Nacional Aut´onoma de M´exico, located in the central highland of M´exico, at 1800 m, with a semi-dry temperature climate. Group F: Six cycling crossbred goats (during October, confirmed by serum progesterone concentrations >1 ng/ml and ultrasonography) of age between 2 and 6 years, mean ± S.D. weight 45 ± 5 kg and a body condition score of 3.0 (Delavaud et al., 2000) were used. Estrus was synchronized using two intramuscular injections 0.075 mg prostaglandin (PG) analogue (Preloban, Intervet, M´exico, Santiago Tianguistenco, M´exico; 0.075 mg/ml prostanglandin) 9 days apart.
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Thirty six hours after the second administration of PG, the goats were observed every 4 h to detect estrus, and once detected, blood samples were collected hourly for the next 24 h. Group A: Four goats in seasonal anestrus (during July, confirmed by serum progesterone concentrations <1 ng/ml and ultrasonography), with similar characteristics as far as breed, age, weight and body condition to those used for the follicular phase portion of the study were utilized. Each goat received 100 g of GnRH (Fertagyl, Intervet-M´exico, 0.1 mg/ml) i.v. to increase the circulating LH concentration. Two blood samples were collected with an interval of 1 h between them before GnRH administration; thereafter, samples were collected every 15 min for 5 h. 2.2. Serum samples for chromatofocusing The LH concentrations in sera collected, allowed the individual identification of the preovulatory peak during the follicular phase, and of that induced during anestrus. For the analysis of LH heterogeneity, only the samples with the maximum concentrations in the pre-ovulatory peak or at 150 min after the administration of 100 g i.v. of GnRH per animal were used. The selected samples (4 ml of serum each) were transferred to dialysis membranes (molecular weight cut-off, 12,000–14,000; Spectrum Medical Industries, Los Angeles, CA, USA) and dialyzed at 4 ◦ C for 48 h with successive change of deionized water every 8 h. Dialyzed serum was lyophilized. 2.3. Chromatofocusing Luteinizing hormone isoforms present in serum were separated according to their charge as previously described (Grotjan and Zalesky, 1991; Perera-Mar´ın et al., 2005). Briefly, the selected sample dialyzed and lyophilized (20 mg of protein) was re-suspended in 4 ml Pharmalyte (Pharmacia Biotech, Uppsala, Sweden) (pH 8.0–10.5)–HCl, diluted 1:45 with deionized water, adjusted to pH 7.0 with 5N HCl and applied to a 27 cm × 0.7 cm (i.d.) column pre-packed with an ionicexchange resin (PBE-118), previously balanced with 20 volumes of 0.025 M HCl–triethylamine (pH 11.0) and kept at 4 ◦ C. Eluted fractions (2 ml) were collected with a flow of 7 ml/h. The pH of each fraction was measured, and when pH 7.0 was reached and stable for more than 10 fractions, the elution buffer was changed for polybuffer 74 (Pharmacia, Biotech), diluted 1:8 with deionized water (pH 3.5) with the aim to obtain the proteins eluted at pH 7.0–3.5. Proteins bound to the column after elution at the lower pH (pH 3.5) were recovered by addition of 1.0 M NaCl to the chromatographic column. Fractions eluted with the pharmalyte buffer were neutralized with a 1.1 M Tris–HCl solution, pH 7.4 (200 l), while fractions eluted with polybuffer 74 and NaCl were neutralized with 1.1 M imidazole, pH 7.0 (200 l). All chromatographic techniques were performed with degasified buffers. The LH concentration was determined in each of the fractions subjected to pH neutralization by radioimmunoassay (RIA), as described in the next section. 2.4. Luteinizing hormone radioimmunoassay for serum samples Luteinizing hormone concentration of each of the serum samples collected during the follicular phase and anestrus, as well as in the fractions obtained during chromatofocusing, were quantified in triplicate with a liquid phase RIA system, specific for LH, with 120 h of incubation at 4 ◦ C (Perera-Mar´ın et al., 2005). Briefly: the RIA was developed using NIDDK-oLH-I-2 as tracer, labeled with the IODO-GEN technique (Arrieta et al., 2006). The LH reference preparation corresponded to NIDDK-oLH-I-2 at doses of 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10 ng per tube, and the first antibody (anti-oLH-26) was used at a final dilution of 1:200,000 (after characterization
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and titration). The separation of the bound fraction from the free fraction was performed with a second antibody (donkey anti-rabbit IgG), diluted 1:80, after and incubation at 4 ◦ C for 24 h. The RIA buffer consisted of phosphates (0.05 M, pH 7.2), 0.14 M NaCl and 0.1% (wt./vol.) bovine serum albumin (BSA). The assay sensitivity was 0.03 ng per tube and the intra- and interassay coefficients of variation corresponded to 6.4% and 10.3%, respectively. Evaluation of the specificity of the system has been previously reported (Arrieta et al., 2006). 2.5. Statistics The percentage of LH eluted in each fraction was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic forms (pH ≤ 6.4) as well as into eight ranges of elution pH (pH 3.5–3.99; 4.9–4.99; 5.0–5.99; 6.0–6.99; 7.0–7.99; 8.0–8.99; 9.0–9.99 and ≥10). To normalize the data, the percentage of recovered immunoreactive LH per each group of pH and pH unit were transformed into arc sine (arc sine of the square root of the proportion, corrected to an angle between 0◦ and 90◦ ) and subjected to analysis of variance (ANOVA), considering the effects of the pH group and the physiological status of the goat sampled (follicular phase or anestrus). The statistical analysis was performed with the SAS (Statistical Analysis System) statistics software. 3. Results The LH secretion (mean ± standard error) is depicted in Fig. 1 for the goats during the follicular phase (panel a) and anestrus (panel b). The LH concentration before GnRH administration (group A) was 0.3 ± 0.03 ng/ml, while 30 min after, LH concentration increased to values of 5 ± 2 ng/ml reaching a zenith at 2.5 ± 0.7 h (10 ± 1 ng/ml). The goats in group F showed the pre-ovulatory LH peak at 7 ± 2 h after the detected estrus, with a circulating concentration of 23 ± 10 ng/ml. The elution patterns (mean ± standard error) of sera analyzed with chromatofocusing are depicted in Fig. 2. Analysis by pH unit (Fig. 3) showed a very basic elution isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during the anestrus, and 3 ± 1% during follicular phase (p < 0.05). Predominant isoforms in the anestrus eluted in the pH range 9.99–9.0 and represented a percentage of 42 ± 3%; in the follicular phase it was of 7 ± 3% (p < 0.01). In group F the predominant isoforms eluted in the pH range 8.99–8.0 (55 ± 8%), while in group A the percentage in that pH range was of 11 ± 2% (p < 0.01). Isoforms eluted in the range of pH 7.99–7, were in a significantly more abundant during anestrus (5.0 ± 0.6%) as compared with the follicular phase (3 ± 1%). The percentage of isoforms that eluted between 6.99 and 3.5 of pH were similar in the two physiological conditions (p > 0.05). When isoforms were grouped by basic, neutral and acidic, the analysis (Fig. 4) indicated that there were not significant differences (p > 0.05) in the proportion of basic and acidic isoforms between the anestrous and follicular phases. The basic forms were the most abundant (64 ± 3%; 68 ± 7%), followed by acidic forms (25 ± 2%; 27 ± 7%). The neutral forms, which eluted in more restricted pH range (7.4–6.5), were in the lesser proportion both in the anestrous and follicular phases (12 ± 0.8%; 5 ± 2%, p < 0.05, respectively). 4. Discussion There were no previous studies found on the pattern of heterogeneity of the LH isoforms in the serum of goats during different physiological conditions; the polymorphism of this hormone in that species has only been reported in the pituitary gland (Perera et al., 1996).
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Fig. 1. Distribution profile of goat serum LH obtained during the follicular phase (panel a) and anestrus (panel b). Each graph shows the LH profile mean ± standard error. The samples used for analysis of LH heterogeneity by chromatofocusing corresponded to the zenith of the pre-ovulatory LH peak or at 150 min after the administration of 100 g i.v. of GnRH per animal. The samples obtained from each animal were collected every 1 h after detection of estrous through 24 h post-estrous, and at 15 min intervals for 5 h after administration of GnRH in the anestrous.
The present study analyzed the serum LH isoforms distribution in two physiological conditions (anestrus and follicular phase) in the goat, characterized by different regulation mechanisms and patterns of secretion (Caraty et al., 1989; Karsch et al., 1993; Caraty and Skinner, 1999). In both physiological conditions, the qualitative chromatofocusing elution pattern of circulating LH isoforms was similar, which coincides with previous observations in the bovine (Perera-Mar´ın et al., 2005) and ovine (Arrieta et al., 2006) species sera. In this study, the total amount of LH in each sample was considered only that contained in the fractions that eluted before the ionic force (1 M NaCl) was applied to the chromatographic column, which differs with other studies that also considered as LH that measured after NaCl (Castro-Fern´andez et al., 2000; Padmanabhan et al., 1992; Hassing et al., 1993). The reason for this decision was based on the demonstration that the protein eluting after 1 M NaCl passes trough
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Fig. 2. Distribution pattern of LH during chromatofocusing separation of sera obtained during the follicular phase (panel a) or anestrus (panel b) of goats. Detailed patterns of LH distribution mean ± standard error are depicted. Twenty milligrams of protein were applied to the ionic exchange column (PBE-118), balanced with 0.025 M triethylamine, pH 11.0, and the column was eluted with a pH gradient of 10–3.5 and 160 (2 ml) fractions were collected. The arrows indicate the time at which the buffer changed during the chromatographic evaluation and after application of 1 M NaCl.
the column, is collected in a solution that alters the antigen–antibody reaction, leaving a great amount of labeled hormone as free, which is erroneously interpreted as a displacement by the endogenous hormone (Perera-Mar´ın et al., 2005). Another methodological aspect that deserves some discussion is that related to the manner the physiological conditions studied were sampled: the anestrus is characterized by LH pulses of low frequency and high amplitude, and lesser mean serum concentrations of LH owing to the negative feedback imposed by estradiol on the secretion of this hormone (Caraty et al., 1989; Karsch et al., 1993). An examination of the relative abundance of different circulating LH isoforms during anestrus would have not been feasible due to the sensibility of the current methodology. Therefore, a single dose of GnRH was administered to provoke a peak of circulating LH during anestrus to be able to have enough LH in the serum to divide it into 160 fractions with measurable quantities in each of the fractions.
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Fig. 3. Percentage of distribution of circulating LH isoforms in goats by unit pH. Results show the percentage ± standard error of each group of isoforms. Serum samples during the estrous and anestrus were analyzed by chromatofocusing, eluted at pH range of 10.5–3.5. For the anestrus, samples were those collected 150 min after 100 g of i.v. GnRH, while the samples collected during estrous were those collected during the zenith of the pre-ovulatory LH peak. The notations a and b denotes differences (p < 0.05) between phases.
It may be argued that the administration of a single i.v. dose of GnRH may modify the characteristics of the LH commonly secreted during anestrus, which cannot be discarded if GnRH induces de novo synthesis in the gonadotroph in a very short time. However, although it has been documented that pulsatile infusion of GnRH analogues increases the proportion of basic serum LH isoforms (Wide and Bakos, 1993) and the transcription of the gene for LH (Burger et al., 2002), studies using a single dose of GnRH in vitro (Zaleky and Grotjan, 1991) and in vivo (Zambrano et al., 1995; Castro-Fern´andez et al., 2000; Perera-Mar´ın et al., 2005; Arrieta et al., 2006) found that there were no changes in the pattern of secreted isoforms of LH. In ovine
Fig. 4. Percentage of distribution of circulating LH isoforms in goats by group pH. Results show the percentage ± standard error of each group of isoforms. Serum samples during the estrous and anestrus were analyzed by chromatofocusing, eluted at pH range of 10.5–3.5. For the anestrus, samples were those collected 15 min after 100 g of i.v. GnRH, while the samples collected during estrus were those collected during the zenith of the pre-ovulatory LH peak. The notation a and b denotes differences (p < 0.05) between phases.
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pituitaries, Brown and McNeilly (1999) determined that, under the stimulus of a single dose of GnRH in the luteal phase, the mRNA for the -subunit of LH was unstable. Additional evidence revealed that the administration of an i.v. bolus of GnRH at diestrus did not increase the mRNA for the GnRH receptor in the gonadotroph (Cowley et al., 1998). This indicates that the acute effect of the GnRH hardly involves de novo LH synthesis, suggesting that, at this stage, GnRH only participates in the release of LH already present in the secretory granules of the gonadotrophs. Therefore, the results of the present study allow us to infer that serum LH analysed during the anestrus of the caprine species, under the experimental conditions imposed, basically correspond to the preformed hormone with the features of the hormone physiologically secreted at this stage. The presence of greater relative amounts of more basic isoforms during anestrous condition as compared with the pre-ovulatory peak, is in agreement with other reports where under low estradiol concentrations, there is a larger proportion of basic LH isoforms. This has been observed in both serum (Perera-Mar´ın et al., 2005; Arrieta et al., 2006) and the pituitary gland (PereraMar´ın, 2003; Arrieta et al., 2006) during the bovine and ovine luteal phases; in pituitary extracts of gonadectomized rams (Christianson et al., 1998) and in ovine and bovine females immunized with antibodies against GnRH (Stumpf et al., 1992; Zalesky et al., 1993). This may indicate that the percentage of basic isoforms in the pituitary and serum increases when the estradiol/progesterone ratios are small (Ulloa-Aguirre et al., 1992) or there is a decreased secretion of GnRH. In contrast, in the follicular phase, including the pre-ovulatory peak, the distribution of LH isoforms tended to be less basic. The physiological importance of the relative abundance of the different isoforms of LH is related to their differences in biological activity. Norris et al. (1989), found that the in vitro biological activity of serum LH in estrous ewes was less when compared with LH during the other phases of the estrous cycle. On the other hand, the in vivo activity is higher for the more acidic isoforms (Fiete et al., 1991) because their clearance rate from circulation is slower (Drickamer, 1991; Baezinger et al., 1992) and because there are differences in the interaction with the specific receptors, depending on the structure of the carbohydrates of the LH molecule. Estradiol participation in gonadotropin glycosylation has been documented. For example, the estradiol modifies in various ways the regulation of gonadotropins’ synthesis and secretion (Ulloa-Aguirre et al., 1988, 1995; Robertson et al., 1991; Ropelato et al., 1999): influencing GnRH synthesis and frequency of secretion (Gharib et al., 1990; Cowley et al., 1998; Nett et al., 2002); inducing the synthesis and quantity of mRNA that codes for the ␣ and LH subunits (Gharib et al., 1990; Hamernik et al., 1995; Di Gregorio and Nett, 1995); regulating glycosylation in the follicular phase, to decrease the activity of N-acetylgalactosamine transferase and GalNac-4-Osulfotransferase, responsible for the incorporation of galactose and sulfate into the gonadotropin N-linked oligosaccharides (Dharmesh and Baenzinger, 1993; Helton and Magner, 1994; Dami´anMatsumura et al., 1999). On the contrary, greater blood concentrations of progesterone, as those observed during the luteal phase, have an opposite effect that can lead to an increase in the presence, and activity, of enzymes responsible for the incorporation of N-acetylgalactosamine and sulfates to the oligosaccharides residues of LH. This communication contributes evidence of the relative abundance of different LH circulating isoforms during the estrous and anestrous phases in the goat. The LH isoforms tend to be more basic during anestrus and this may be related to the effect of estradiol. Acknowledgements The authors wish to thank the National Council for Science and Technology (CoNaCyT) for financing the project (25748-B). We are also grateful to the National Institute of Health (NIH) for
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the donation of hormones for the characterization of isoforms and to the Neurobiology Institute UNAM for the donation of the first antibody used in this study. Susana Rojas Maya is a graduate student at UNAM. Isabel P´erez Montfort translated the manuscript to English. References Arrieta, E., Porras, A., Gonz´alez-Padilla, E., Murcia, C., Rojas, S., Perera-Mar´ın, G., 2006. Ovine serum and pituitary isoforms of luteinising hormone during the luteal phase. Reprod. Fert. Develop. 18, 485–495. Baenzinger, J.U., Green, E.D., 1988. Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim. Biophys. Acta 947, 287–306. Baezinger, U.J., Kumar, S., Brodbeck, R.M., Smith, P.L., Beranek, M.C., 1992. Circulatory half-life but not interaction with the lutropin/chorionic gonadotropins receptor is modulated by sulfation of bovine lutropin oligosaccharides. Proc. Natl. Acad. Sci. USA 89, 334–338. Barrios-De-Tomasi, J., Timosi, C., Merchant, H., Quintanar, A., Av´alos, J.M., Andersen, C.Y., Ulloa-Aguirre, A., 2002. Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones. Mol. Cell. Endocrinol. 186, 189–198. Brown, P., McNeilly, A.S., 1999. Transcriptional regulation of gonadotropin genes. Rev. Reprod. 4, 117–124. Burger, L.L., Dalkin, A.C., Aylor, K.W., Haisenleder, D.J., Marshall, J.C., 2002. GnRH pulse frequency modulation of gonadotropin subunit gene transcription in normal gonadotropes-assessment by primary transcript assay provides evidence for roles of GnRH and follistatin. Endocrinology 143, 3243–3249. Burgon, P.G., Stanton, P.G., Robertson, D.M., 1996. In vivo bioactivities and clearance patterns of highly purified human luteinizing hormone isoforms. Endocrinology 137, 4827–4836. Caraty, A., Locatelli, A., Martin, G.B., 1989. Biphasic response in the secretion gonadotrophin-releasing hormone in ovariectomized ewes injected with estradiol. J. Endocrinol. 123, 375–382. Caraty, A., Skinner, D.C., 1999. Progesterone priming is essential for the full expression of the positive feedback effect of estradiol in inducing the preovulatory gonadotropin-releasing hormone surge in the ewe. Endocrinology 140, 165–170. Castro-Fern´andez, C., Olivares, A., Soderlund, D., L´opez-Alvarenga, J.C., Zambrano, E., Veldhuis, J.D., Ulloa-Aguirre, A., M´endez, J.P., 2000. A preponderance of circulating basic isoforms is associated with decreased plasma half-life and biological to immunological ratio of gonadotropin-releasing hormone-releasable luteinizing hormone in obese men. J. Clin. Endocrinol. Metab. 85, 4603–4610. Chemineau, P., Martin, G.B., Saumande, J., Normant, E., 1988. Seasonal and hormonal control of pulsatile LH secretion in the dairy goat (Capra hircus). J. Reprod. Fert. 83, 91–98. Christianson, S.L., Zalesky, D.D., Grotjan, H.E., 1998. Ovine luteinizing hormone heterogeneity: androgens increase the percentage of less basic isohormone. Domest. Anim. Endocrinol. 15, 87–92. Combarnous, Y., 1988. Stucture and structure–function relationships in gonadotropins. Reprod. Nutr. Develop. 28, 211–228. Cooke, D.J., Crowe, M.A., Roche, J.F., Headon, D.R., 1996. Gonadotrophin heterogeneity and its role in farm animal reproduction. Anim. Reprod. Sci. 41, 77–99. Cooke, D.J., Cowley, M.A., Roche, J.F., 1997. Circulating FSH isoforms patterns during recurrent increases in FSH throughout the oestrus cycle of heifers. J. Reprod. 110, 339–345. Cowley, M.A., Rao, A., Wright, P.J., Illing, N., Millar, R.P., Clarke, I.J., 1998. Evidence for differential regulation of multiple transcripts of the gonadotropin releasing hormone receptor in the ovine pituitary gland; effect of estrogen. Mol. Cell. Endocrinol. 146, 141–149. Creus, S., Chaia, Z., Pellizzari, E.H., Cigorraga, S.B., Ulloa-Aguirre, A., Campo, S., 2001. Human FSH isoforms: carbohydrate complexity as determinant of in-vitro bioactivity. Mol. Cell. Endocrinol. 174, 41–49. Cupp, A.S., Kojima, F.N., Roberson, M.S., Stumpf, T.T., Wolfe, M.W., Werth, L.A., Kittok, R.J., Grotjan, H.E., Kinder, J.E., 1995. Increasing concentration of 17-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. Biol. Reprod. 52, 288–296. Dami´an-Matsumura, P., Zaga, V., Maldonado, A., S´anchez-Hern´andez, C., Timossi, C., Ulloa-Aguirre, A., 1999. Oestrogens regulate pituitary ␣2,3-sialyltransferase messenger ribonucleic acid levels in the female rat. J. Mol. Endocrinol. 23, 153–165. Delavaud, C., Bocquier, F., Chilliard, Y., Keisler, D.H., Gertler, A., Kann, G., 2000. Plasma leptin determination in ruminants: effect of nutritional status and body fatness on plasma leptin concentration assessed by a specific RIA in sheep. J. Endocrinol. 165, 519–526.
S. Rojas-Maya et al. / Animal Reproduction Science 100 (2007) 280–290
289
Dharmesh, S.M., Baenzinger, J.U., 1993. Estrogen modulates expression of the glycosyltransferases that synthesize sulfated oligosaccharides of lutropin. Proc. Natl. Acad. Sci. USA 90, 11127–11131. Drickamer, K., 1991. Clearing up glycoprotein hormone. Cell 67, 1029–1032. Di Gregorio, G.B., Nett, T., 1995. Estradiol and progesterone influence the synthesis of gonadotropins in the absence of gonadotropin-releasing hormone in the ewe. Biol. Reprod. 53, 166–172. Fiete, D., Srivastava, V., Hindsgaul, O., Baenzinger, J.U., 1991. A hepatic reticuloendothelial cell receptor specific for SO4 -4GalNAc beta1, 4GlcNAc beta1, 2Man alpha that mediates rapid clearance of lutropin. Cell 67, 1103–1110. Gharib, S.D., Wierman, M.E., Shupnik, M.A., Chin, W.W., 1990. Molecular biology of the pituitary gonadotropins. Endocr. Rev. 11, 177–198. Grotjan, H.E., Zalesky, D.D., 1991. Ovine luteinizing hormone VI. Analysis of the misclassification errors in the separation of intrapituitary isohormones by chromatofocusing. J. Chromatogr. A 549, 153–158. Hamernik, D.L., Clay, C.M., Turzillo, A., Van Kirk, E.A., Moss, G.E., 1995. Estradiol increases amounts of messenger ribonucleic acid for gonadotropin-releasing hormone receptors in sheep. Biol. Reprod. 53, 179–185. Hassing, J.M., Kletter, G.B., I’Anson, H., Wood, R.I., Beitins, I.Z., Foster, D.L., Padmanabhan, V., 1993. Pulsatile administration of gonadotropin-releasing hormone does not alter the follicle-stimulating hormone (FSH) isoforms distribution pattern of pituitary or circulating FSH in nutritionally growth-restricted ovariectomized lambs. Endocrinology 132, 1527–1536. Helton, T.E., Magner, J.A., 1994. Sialytransferase messenger ribonucleic acid increases in thyrotrophs of hypothyroid mice: an in situ hibridization study. Endocrinology 134, 2347–2352. Karsch, F.J., Dahl, G.E., Evans, N.P., Manning, J.M., Mayfiel, K.P., Moenter, S.M., Foster, D.L., 1993. Seasonal changes in gonadotropin-releasing hormone secretion in the ewe-alteration in response to the negative feedback action of estradiol. Biol. Reprod. 49, 1377–1383. Keel, B.A., Schanbacher, B.D., Grotjan Jr., H.E., 1987. Ovine luteinizing hormone. I. Effect of castration and steroid administration on the charge heterogeneity of pituitary luteinizing hormone. Biol. Reprod. 36, 1102–1113. Kojima, F.N., Cupp, A.S., Stumpf, T.T., Zalesky, D.D., Roberson, M.S., Werth, L.A., Wolfe, M.W., Kittok, R.J., Grotjan, H.E., Kinder, J.E., 1995. Effects of 17 -estradiol on distribution of pituitary isoforms of luteinizing hormone and follicle-stimulating hormone during the follicular phase of the bovine estrous cycle. Biol. Reprod. 52, 297–304. Legan, J.S., Karsch, J.F., 1979. Neuroendocrine regulation of the estrous cycle and seasonal breeding in the ewe. Biol. Reprod. 20, 74–85. Manzella, S.M., Hooper, L.V., Baenzinger, J.U., 1996. Oligosaccharides containing 1,4-linked N-acetylgalactosamine, a paradigm for protein-specific glycosylation. J. Biol. Biochem. 271, 12117–12120. Mi, Y., Shapiro, S.D., Baenzinger, J.U., 2002. Regulation of lutropin circulatory half-life by the mannose/Nacetylgalactosamine-4-SO4 receptor is critical for implantation in vivo. J. Clin. Invest. 109, 269–276. Nett, T.M., Turzillo, A.M., Baratta, M., Rispoli, L.A., 2002. Pituitary effects of steroid hormones on secretion of folliclestimulating hormone and luteinizing hormone. Domest. Anim. Endocrinol. 23, 33–42. Norris, T.A., Weesner, G.D., Shelton, J.M., Harms, P.G., Forrest, D.W., 1989. Biological activity of LH during the peripartum period and the estrous cycle of the ewe. Domest. Anim. Endocrinol. 6, 25–33. Padmanabhan, V., Mieher, C.D., Borondy, M., I‘Anson, H., Wood, R.I., Landefeld, T.D., Foster, D.L., Beitins, I.Z., 1992. Circulating bioactive follicle-stimulating hormone and less acidic follicle-stimulating hormone isoform during experimental induction of puberty in the female lamb. Endocrinology 131, 213–220. Padmanabhan, V., Lee, J.S., Beitins, I.Z., 1998. Follicle-stimulating isohormones: regulation and biological significance. In: Thacher, W.W., Inskeep, E.K., Niswender, R.D., Doborska. C. (Eds.), Reproduction in domestic ruminants. J. Reprod. Fert. 54, 87–99. Perera, M.G., Ortiz, R.F., Gamboa, V.J.J., Reynoso, M.W., Falc´on, A.A., Salas, V.A., 1996. Obtenci´on, purificaci´on y caracterizaci´on de dos formas de hormona luteinizante de la adenohip´ofisis caprina (gLH). Vet. Mex. 27, 1–10. Perera-Mar´ın, G., 2003. Patr´on de distribuci´on de isoformas de la hormona luteinizante (LH) intrahipofisaria y serica en la especie bovina. PhD. Thesis, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´onoma de M´exico, M´exico D.F. Perera, M.G., Falc´on, A.A., Murcia, M.C., Hern´andez, C.J., Gonz´alez, P.E., 2004. Purification of five bovine luteinizing hormone (bLH) isoforms. Chemical, physical, biological and immunological characterization. Vet. Mex. 35, 129–145. Perera-Mar´ın, G., Murcia, C., Rojas, S., Hern´andez-Cer´on, J., Gonz´alez-Padilla, E., 2005. Pattern of circulating luteinizing hormone isoforms during the estrous and luteal phases in Holstein heifers. Anim. Reprod. Sci. 86, 53–69. Robertson, D.M., Foulds, L.M., Fry, R.C., Cummins, J.T., Clarke, I., 1991. Circulating half-lives of follicle-stimulating hormone and luteinizing hormone in pituitary extracts and isoforms fractions of ovariectomized and intact ewes. Endocrinology 129, 1805–1813.
290
S. Rojas-Maya et al. / Animal Reproduction Science 100 (2007) 280–290
Ropelato, M.G., Garc´ıa-Rudaz, M.C., Castro-Fern´andez, C., Ulloa-Aguirre, A., Escobar, M.E., Barontini, M., Veldhuis, J.D., 1999. A Prepronderance of basic luteinizing hormone (LH) Isoforms accompanies inappropriate hypersecretion of both basal and pulsatile LH in adolescents with polycystic ovarian s´ındrome. J. Clin. Endorcinol. Metab. 84, 4629–4636. Stumpf, T.T., Roberson, M.S., Wolfe, M.W., Zalesky, D.D., Cupp, A.S., Werth, L.A., Kojima, N., HejL, K., Kittok, R.J., Grotjan, H.E., Kinder, J.E., 1992. A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer. Biol. Reprod. 46, 442–450. Tanaka, T., Takanori, O., Hopshino, K., Mori, Y., 1995. Changes in the gonadotropin-releasing hormone pulse generator activity during the estrous cycle in the goat. Neuroendocrinology 62, 553–561. Ulloa-Aguirre, A., Espinoza, R., Dami´an-Matsumura, P., Larrea, F., Flores, A., Morales, I., D´ominguez, R., 1988. Studies on the microheterogeneity of anterior pituitary follicle-stimulating hormone in the female rat. Isoelectric pattern throughout the estrous cycle. Biol. Reprod. 38, 70–78. Ulloa-Aguirre, A., Cravioto, A., Damian-Matsumura, P., Jimenez, M., Zambrano, E., D´ıaz-S´anchez, V., 1992. Biological characterization of the natural ocurring analogoues of intrapituitary human follicle-stimulating hormone. Hum. Reprod. 7, 23–30. Ulloa-Aguirre, A., Midgley, A.R., Beitins, I.Z., Padmanabhan, V., 1995. Follicle-stimulating isohormones: characterization and physiological relevance. Endocr. Rev. 16, 765–787. Ulloa-Aguirre, A., Timossi, C., Dami´an-Matsumura, P., Dias, J.A., 1999. Role of glycosylation in function of folliclestimulating hormone. Endocrine 11, 205–215. Wide, L., Bakos, O., 1993. More basic forms of both human follicle-stimulating hormone and luteinizing hormone in serum at midcycle compared with the follicular or luteal phase. J. Clin. Endocrinol. Metab. 76, 885–889. Zaleky, D.D., Grotjan, H.E., 1991. Comparison of intracellular and secreted isoforms of bovine and ovine luteinizing hormone. Biol. Reprod. 4, 1016–1024. Zalesky, D.D., Nett, T.M., Grotjan, H.E., 1992. Ovine luteinizing hormone: isoforms in the pituitary during the follicular and luteal phases of the estrous cycle and during anestrous. J. Anim. Sci. 70, 3851–3856. Zalesky, D.D., Schanbacher, B.D., Grotjan, H.E., 1993. Effect of immunization against LHRH on isoforms of LH in the ovine pituitary. J. Reprod. Fert. 99, 231–235. Zambrano, E., Olivares, A., M´endez, J.P., Guerrero, L., D´ıaz-Cueto, L., Veldhuis, J.D., Ulloa-Aguirre, A., 1995. Dynamics of basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoforms distribution throughout the human menstrual cycle. J. Clin. Endocrinol. Metab. 80, 1647–1656.