Biomedical subjects

J F Roser

Publications and source records attributed to J F Roser.

At least 19 recordsLinked to original sources

PubMed2005

Reducing estrogen synthesis does not affect gonadotropin secretion in the developing boar.

Boars have high concentrations of plasma and testicular estrogens, but how this hormone is involved in feedback regulation of the gonadotropins and local regulation of testicular hormone production is unclear. The present study examined the effects of reducing endogenous estrogens by aromatase inhibition on concentrations of plasma LH and FSH and on testicular and plasma concentrations of testosterone (T) and immunoreactive inhibin (INH). Thirty-six littermate pairs of boars were used. One boar from each pair was assigned to the control group (vehicle); the other boar to the treatment group (aromatase enzyme inhibitor, Letrozole, 0.1 mg/kg body weight [BW]). Weekly oral treatment started at 1 wk of age and continued until castration at 2, 3, 4, 5, 6, 7, or 8 mo. Plasma concentrations of gonadotropins, INH, T, estradiol (E2), and estrogen conjugates (ECs) were determined. Testicular tissue was collected at castration for determination of INH and T and for confirmation of reduced aromatase activity. The acute effects of aromatase inhibition on gonadotropins were monitored in two adult boars treated once with Letrozole (0.1 mg/kg BW). Treatment with the aromatase inhibitor reduced testicular aromatase activity by 90% and decreased E2 and ECs without changing acute, long-term, or postcastration LH and FSH. Plasma T, testicular T, and circulating INH concentrations did not change. Testicular INH was elevated in treated boars compared with controls. In conclusion, estrogen does not appear to play a regulatory role on gonadotropin secretion in the developing boar. This is in direct contrast to findings in males of several other species.

Animals↗

PubMed2005

Dopamine receptors in equine ovarian tissues.

Dopamine (DA) agonist and antagonist treatments can affect ovarian reproductive events in the mare. To support our theory that DA produces these effects by acting directly on the ovary, we analyzed equine ovarian tissues for the presence of dopamine receptor-1 (D1r) and dopamine receptor-2 (D2r) mRNA by reverse transcription polymerase chain reaction (RT-PCR) and D1r and D2r proteins by Western blot and immunohistochemistry (IHC). RT-PCR was performed on RNA isolated from ovarian cortex, medulla, granulosa/theca or corpus luteum (CL) tissues and from pituitary (D2r control) and renal artery (D1r control). D1r and D2r specific primers were designed from partial DNA sequences known for the horse (D2r) or conserved sequences from other species (D1r). Western blot analyses were conducted on CL, cortex and granulosa/theca samples and IHC was performed on CL tissues using D1r or D2r specific antibodies. The incidence of positive D2r mRNA was high in CL and ovarian cortex, low in granulosa/theca, and not detectable in ovarian medulla. Dopamine D1r mRNA incidence was high (50%) only in CL tissues. D1r and D2r antibody staining was positive for each tissue type analyzed by Western blot procedures. All CL tissues prepared by IHC showed positive staining for D1r and D2r proteins. Both DA receptor proteins appeared uniformly distributed throughout the CL tissue. These results indicate that equine ovarian tissues do possess D1r and D2r, and suggests that DA can act directly on ovarian tissues through its interaction with DA receptors.

Animals↗

PubMed2001

Endocrine and paracrine control of sperm production in stallions.

The specific nature and relative contribution of the major hormones involved in regulation of reproductive function of the stallion are not well defined nor have paracrine or autocrine factors been identified. Over the last 12 years, our laboratory has been engaged in characterizing the hypothalamic-pituitary-testicular axis (HPT) in stallions. A number of endocrine factors and mechanisms important for normal reproductive function have been investigated. Studies investigating poor fertility in stallions suggest that a closer look at the testicular level is warranted. For a complete understanding of intratesticular control mechanisms including cell-to-cell interactions in the stallion, studies on the actions of paracrine/autocrine factors such as growth factors, inhibin, activin, and oxytocin are needed. In other species, paracrine/autocrine systems appear to be important in modulating endocrine control of testicular function and spermatogenesis.

Animals↗

PubMed2001

The effects of age, season and fertility status on plasma and intratesticular insulin-like growth factor I concentration in stallions.

The purposes of this study were to establish the basal plasma and testicular insulin-like growth factor-I (IGF-I) values for stallions ranging in age from 6 months to 23 years and to determine if IGF-I could be used as a marker for declining fertility. Blood and testes were obtained from 28 light horse stallions and colts. Of the 28 stallions, 22 were considered fertile and were categorized by age (<2 y, 5 to 10 y, 11 to 15 y, and 16 to 23 y); 12 age-matched stallions were grouped as to fertility status (fertile, subfertile, infertile); and all 28 stallions were grouped as to season of castration (breeding season vs. non-breeding season). In colts less than 2 years of age, IGF-I concentrations in plasma and testicular extracts were higher (P < 0.01) than in the other age groups and were higher in the breeding season than in the non-breeding season (P < 0.01). No significant differences in plasma or testicular extract concentrations of IGF-I were found among fertility groups. The results of this study demonstrate that plasma and testicular IGF-I levels are high in stallions younger than 2 years of age and then decline and plateau in stallions older than 5 years of age, suggesting that IGF-I may be involved in testicular development. The results allude to a possible seasonal effect on IGF-I production. However, it is difficult to separate true seasonality and the effect of age as only those stallions less than 2 years old exhibited variation between seasons. The IGF-I does not appear to have a direct relationship with declined fertility in the stallions tested, suggesting that IGF-I may not be a reliable biomarker for the diagnosis of subfertility and infertility.

Aging↗

PubMed2001

Maternal-conceptus signalling during early pregnancy in mares: oestrogen and insulin-like growth factor I.

Embryonic production of oestrogen is thought to play an important role in conceptus-maternal signalling during early pregnancy in mares, and may be regulated in an autocrine or paracrine fashion by insulin-like growth factor I (IGF-I). In this study, the hypothesis that IGF-I stimulates embryonic oestrogen synthesis, which in turn stimulates uterine IGF-I secretion was tested. Specific sources of IGF-I in the uterine lumen were characterized. Preimplantation embryos, uterine biopsies, and uterine flush fluids were collected on day 13 of pregnancy. Embryos were cultured whole for 24 h, or dispersed and incubated in serum-free culture medium supplemented with androstenedione or testosterone (0-10 microg ml(-1)) and IGF-I (0-100 microg ml(-1)). Oestrogen synthesis was increased by addition of androgen, but there was no dose-dependent effect of IGF-I. Endometrial explants were cultured for 24, 48 and 72 h in serum-free medium supplemented with oestradiol. IGF-I was measured by radioimmunoassay in embryo-conditioned medium, explant culture medium, blastocoelic fluid, concentrated (x 100) uterine flush fluid and endometrial-tissue homogenate. Both the embryo and endometrium produced significant quantities of IGF-I, indicating a role for this growth factor in autocrine-paracrine signalling during early pregnancy. However, secretion of IGF-I by endometrial explants was not modulated by oestrogen.

Animals↗

PubMed2000

Tissue-specific localization of cytochrome P450 aromatase in the equine embryo by in situ hybridization and immunocytochemistry.

Estrogen production by the preimplantation equine embryo is presumed to be important in maternal-conceptus communication in the mare. The synthesis of C(18) estrogens from C(19) androgens requires cytochrome P450 aromatase (P450(arom)) in the conceptus, but little information is available on the specific tissue location or potential developmental patterns of expression for the horse. The goal of this research was to localize P450(arom) in the equine conceptus by immunocytochemistry and in situ hybridization. Intact blastocyst-stage embryos were collected by nonsurgical flush on Days 12-15 of pregnancy, fixed in 4% paraformaldehyde, and paraffin-embedded. Aromatase protein was localized using rabbit anti-human placental aromatase antiserum with a detection system utilizing peroxidase and 3-amino-9-ethylcarbazole. For in situ hybridization, tissue sections were incubated with sense or antisense [(35)S]UTP-labeled cRNA probes prepared from equine aromatase cDNA. Aromatase protein and transcript were abundant in the extraembryonic trophectoderm but absent from embryonic ectoderm. No P450(arom) expression was detected in abembryonic endoderm or mesoderm. Aromatase expression was demonstrated in the endoderm beneath the disc (hypoblast). This pattern of P450(arom) expression in the equine blastocyst closely resembles that seen transiently in the porcine embryo, suggesting that regulatory mechanisms conferring tissue specificity may be conserved.

Animals↗

PubMed2000

Oxytocin release and its relationship to dihydro-15-keto PGF2alpha and arginine vasopressin release during parturition and to suckling in postpartum mares.

Pituitary blood was collected from the intercavernal sinus in five mares before and during parturition, and in nine mares immediately after parturition to investigate oxytocin patterns during parturition and early lactation, and to determine the relationship between oxytocin, prostaglandin and arginine vasopressin during parturition. In four mares in which sample collection began at least 6 h before rupture of the chorioallantois, a significant increase (P < 0.05) in PGF(2alpha) concentration was detected before a significant increase in oxytocin concentration. Cross-correlation analysis of log-transformed oxytocin and PGF(2alpha) concentrations revealed a significant correlation (P < 0.05) at a 6 min lag period, indicating that in the 2 h before delivery of the foal, an increase in prostaglandin was followed 6 min later by an increase in oxytocin. A significant effect of suckling on oxytocin release by the mare was detected in only two of nine mares, when oxytocin concentrations were evaluated 0-3 min after suckling. When foals were prevented from sucking for 1 h, by being either muzzled (n = 2) or separated from the mare (n = 2), there was no significant association between resumption of suckling and oxytocin release by the mare. The results of these studies show that: (i) oxytocin secretion from the maternal posterior pituitary gland begins before, or in association with, the onset of the second stage of labour, and that prostaglandin increases in the peripheral circulation before oxytocin release; and (ii) suckling is not significantly related to oxytocin release in mares.

Animals↗

PubMed1998

Effects of age, season, and fertility status on plasma and intratesticular immunoreactive (IR) inhibin concentrations in stallions.

The nature of the relationship between inhibin and reproductive function in the stallion is yet to be elucidated. Blood and testes from 51 light horse stallions ranging in age from 2 mo to 25 years were collected during the breeding and nonbreeding seasons to study the effects of testicular maturation, aging, season, and fertility status on peripheral and intratesticular concentrations of Ir inhibin and other reproductive hormones. Of the 51 stallions, 12 age-matched stallions (6 fertile, 3 subfertile, and 3 infertile) were used in the fertility study. Blood samples were taken before castration and plasma stored at -20 degrees C for analysis of Ir inhibin, luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), estradiol (E2), and estrogen conjugates (EC) by radioimmunoassay (RIA). Testes were homogenized and testicular extracts prepared and frozen at -70 degrees C for analysis of Ir inhibin, T, E2, and EC by RIA. Plasma concentrations of Ir inhibin, LH, FSH, T, E2, and EC and intratesticular concentrations of Ir inhibin, T, E2, and EC increased with age (P < 0.01). The most dramatic effect appeared to be during testicular maturation. An aging effect was not observed in adult stallions. A seasonal effect was not detected for any of the plasma hormones, whereas for the intratesticular hormones the only change noted was an increase in T in the nonbreeding season (P < 0.05). Plasma Ir inhibin, E2, and EC were lower (P < 0.01) and gonadotropins higher (P < 0.05) in infertile stallions. Plasma T levels did not change. Intratesticular Ir inhibin concentrations tended to be lower (P < 0.1) in subfertile stallions and significantly lower (P < 0.01) in infertile stallions, whereas intratesticular steroid levels were not different among the three groups. In conclusion, plasma and intratesticular Ir inhibin concentrations seem to be affected by testicular maturation and fertility status.

Aging↗

PubMed1997

Endocrine basis for testicular function in the stallion.

The specific nature and relative contribution of the various factors involved in the endocrine/paracrine/autocrine control of reproductive function in normal stallions are not well defined nor have they been elucidated in the idiopathic subfertile/infertile stallion. Over the last 9 years, work in our laboratory has been focused in characterizing the hypothalamic-pituitary-testicular axis in fertile, subfertile (idiopathic oligospermia) and infertile (idiopathic azoospermia) stallions. We have identified endocrine factors and mechanisms important for normal reproductive function, and demonstrated specific hormonal changes in pituitary and testicular function between fertile, subfertile and infertile stallions. Recent evidence suggests that the primary defect is at the level of the testes. The nature of the dysfunction does not appear to involve changes in LH receptor binding kinetics but may be related to post-receptor mechanisms.

Journal Article↗

PubMed1997

Production of free estrogens and estrogen conjugates by the preimplantation equine embryo.

In vitro production of free estrogens and estrogen conjugates by intact Day 12.5, 13.5 and 14.5 equine embryos was measured at 2-h intervals over a 24-h culture period. Production of free estrogens was higher for Day 14.5 than Day 12.5 embryos. Differences in production of conjugated estrogens were not significant, but a trend toward increased production with increased age of embryo was apparent. No trend toward increased free and conjugated estrogen production per cell was observed with age. Embryo diameter and number of cells increased with age but varied considerably within groups. The amount of free and conjugated estrogens measured in blastocoelic fluid did not decrease over the 24-h culture period, suggesting that estrogens detected in culture medium were produced by the embryo and not the result of leakage of maternal estrogen from the blastocoele. The results of this study support previous results that estrogen production increases with development of equine embryos. This increase in estrogen production appears to be more closely associated with the diameter of the embryo, and hence its number of cells, than with increased intracellular steroidogenic activity.

Journal Article↗

PubMed1994

Localization of aromatase in equine Leydig cells.

Stallion testes secrete large amounts of estrogens, but the cellular location of the enzyme that converts androgens to estrogens, cytochrome P450 aromatase, has not been determined. The goal of the present study was to immunocytochemically localize stallion testicular aromatase using a polyclonal antibody generated against human placental cytochrome P450 aromatase. Testes were obtained from 12 stallions from 2 to 23 years of age, during both the breeding and non-breeding seasons. Immunoreactivity was confined to the Leydig cells in all testes examined. No immunostaining was observed in the Sertoli or germ cells. Heterogeneity in the level of immunostaining among individual Leydig cells was observed. The results of this study indicate that in postpubertal, adult, and aged stallions, testicular aromatase is located in Leydig cells.

Animals↗

PubMed1994

Inhibin activity in the mare and stallion.

An overnight double antibody RIA, employing a rabbit antiserum raised to bovine 31 kDa inhibin (rAs-#1989, NICHD) and purified bovine 31 kDa inhibin (bINH-I-90/1, NICHD) as trace and standard, was validated to measure immunoreactive inhibin (iINH) concentrations in equine peripheral plasma, follicular fluid (FF), ovarian vein (OV) plasma, testicular tissue extracts (TTE) and testicular vein (TV) plasma. The dynamic relationship of iINH and follicle stimulating hormone (FSH) was investigated during the estrous cycle of the mare and the annual reproductive cycle of the stallion. In the RIA, parallel dose-response curves were observed between the bovine inhibin standard and serial dilutions of equine FF, OV, TTE, TV and plasma. The average recovery of a known amount of purified bovine inhibin added to gelding plasma was approximately 100%. In the inhibin bioassay, serial dilution of equine FF and TTE were observed to be parallel to the bovine inhibin standard. A five-fold difference (p < 0.05) between jugular and gonadal vein plasma iINH concentrations was observed in the mare and an eight-fold difference (p < 0.05) was observed in the stallion. Plasma levels of iINH in ovariectomized mares or geldings were undetectable in the RIA. Concentrations of FSH, estradiol and iINH changed significantly in the mare during the estrous cycle (p < 0.05). Immunoreactive inhibin levels were highest (0.54 +/- 0.06 ng/ml) on the day of ovulation, declined rapidly following ovulation and reached a nadir (0.21 +/- 0.03 ng/ml) on day 7 post-ovulation. Plasma iINH and estradiol concentrations followed a similar profile and were found to be positively correlated (r = 0.7064; p < 0.01), whereas iINH and FSH levels demonstrated an inverse relationship (r = -0.7359, p < 0.01) throughout the estrous cycle. Concentrations of FSH were also inversely related (-0.8498, p < 0.01) with estradiol during the cycle. In the stallion, plasma iINH and FSH levels changed significantly during the year (p < 0.05). The iINH profile reflected seasonal changes in testicular activity, with highest concentrations in late spring (3.37 +/- 0.44 ng/ml) and lowest concentrations in the fall (2.21 +/- 0.33 ng/ml). Plasma concentrations of iINH were positively correlated (r = 0.7691, p < 0.01) with FSH concentrations throughout the year. In conclusion, a specific and sensitive RIA for iINH has been validated for plasma and biological fluids in the horse. Furthermore, the gonads appear to be the source of bioactive and immunoreactive inhibin as observed in other species.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Modulation of gonadotropin-releasing hormone-stimulated luteinizing hormone release in cultured male equine anterior pituitary cells by gonadal steroids.

The objective of the present study was to determine whether the testicular steroids, i.e., testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and estrone sulfate (E1SO4), play a physiological role in regulating LH release in the male horse by direct actions at the anterior pituitary gland. Enzymatically dispersed anterior pituitary cells from stallions (n = 4) or geldings (n = 3) were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse medium. To determine the effects of the steroids on the LH response to GnRH, the cells were incubated for 24 h in fresh media with or without 10(-10) M E2 or 10(-8) M T or DHT followed by a 4-h incubation +/- GnRH (10(-11) to 10(-7) M). Media and cells were analyzed for LH by RIA. In the stallion, GnRH increased LH release (p < 0.001) in a dose-dependent manner (ED50 GnRH = 4.5 x 10(-9) M), and this response was unaltered by T or DHT but greatly enhanced by E2 (p < 0.001). E2 lowered the ED50 for GnRH to 5 x 10(-10) M and increased the maximum LH response to GnRH by 350%. The LH release in response to a constant dose of 1 nM GnRH was unaltered by varying doses of T, DHT, or E1SO4 (10(-11) to 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effect of surgical removal of endometrial cups on concentrations of chorionic gonadotrophin and subsequent fertility in the mare.

Seven pregnant mares underwent general anaesthesia, laparotomy, hysterotomy and removal of a 50-day conceptus. Eversion of the uterine horn through the hysterotomy site allowed direct visualisation and electrosurgical removal of endometrial cup tissue from 5 randomly selected mares (Nos 1-5), while cup tissue in 2 mares (Nos 6 and 7) was left intact. Two pregnant mares served as unoperated controls (Nos 8 and 9). Efforts to re-establish pregnancy were initiated 20 days after surgery. Serum samples collected before surgery and during the post-operative period were analysed for concentration of horse chorionic gonadotrophin (CG) by radioimmunoassay. Mean (+/- sd) weight and area of removed tissue was 9.91 +/- 4.6 g and 22.4 +/- 5.9 cm2, respectively. Concentrations of CG ranging from 3440 to 21,220 ng/ml were highest at the time of cup removal (50 days) and declined thereafter. Peak concentrations of CG at the time of surgery were not linearly correlated (r = -0.59) with mass of excised cup tissue. Individual CG half-life values for Mares 1-5 ranged from 5.4 to 8.8 days (mean 7.1 +/- 1.2 days). Peak CG concentrations occurred 68 days after conception with a rate of disappearance (half-time) of 13.4 +/- 0.4 days in Mares 6 and 7 and at 74 and 78 days after conception with a half-time of 14.0 +/- 5.7 days in Mares 8 and 9. Mares 1-5 exhibited behavioural oestrus and ovulation 30-44 days after surgery. Pregnancy occurred in 3 mares within 41 days after surgery and was associated with concentrations of CG below 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Immunochemical studies of equine chorionic gonadotropin (eCG), eCG alpha, and eCG beta.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

Animals↗

PubMed1993

A supersensitive immunofluorometric assay for rat luteinizing hormone.

We have developed an immunofluorometric assay (IFMA) for rat (r) LH, which is based on two monoclonal antibodies, one to bovine and the other to human LH. Signal detection occurs by time-resolved fluorescence evoked by a europium label (Delfia, Wallac). The method is fast in comparison to the standard RIA with the NIDDK reagents (4 h vs. 3 days). The sensitivity of the IFMA assay (0.75 pg/tube; NIDDK rLH RP-2) is over 30-fold higher than that of the NIDDK RIA (usual detection limit, 20-30 pg/tube). Using 25-microliters serum samples, the sensitivity of IFMA is 0.03 micrograms/liter; with 100-microliters samples, it is 0.0075 micrograms/liter. The cross-reactivity of the IFMA assay is 0.3% with rFSH, 3% with rTSH, and less than 0.05% with rGH, rPRL, and the rat alpha-subunit. A linear correlation between IFMA and RIA values is seen at serum levels above 0.4 micrograms/liter. Below this level, only IFMA is able to detect concentration differences between samples. In practice, this means that only IFMA is able to provide meaningful measurements of suppressed levels of serum LH. The correlation coefficient between IFMA and the mouse interstitial cell in vitro bioassay for LH in randomly selected rat pituitary homogenates was 0.93 (n = 47). The serum concentration of LH determined by IFMA is 0.57 +/- 0.10 micrograms/liter in intact adult male rats (mean +/- SEM; n = 12) and 0.41 +/- 0.10 micrograms/liter (n = 10) in randomly cycling females. The level in hypophysectomized rat serum is 0.035 +/- 0.0033 micrograms/liter (n = 8), if the limit of sensitivity (0.03 microgram/liter) is assigned to unmeasurable levels. One-week treatment of male rats with 2-cm Silastic implants containing testosterone suppressed serum LH, measured by IFMA, from 0.56 +/- 0.057 to 0.086 +/- 0.057 micrograms/liter (P < 0.01). The suppression of LH measured in the same samples by RIA was lower, from 0.73 +/- 0.057 to 0.44 +/- 0.048 micrograms/liter (P < 0.01). A 5-day starvation of intact male rats suppressed serum LH from 0.57 +/- 0.10 to 0.30 +/- 0.05 microgram/liter by IFMA (P < 0.01), whereas the decrease determined by RIA was not significant (0.80 +/- 0.07 vs. 0.66 +/- 0.13 micrograms/liter).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion from perifused equine pituitaries.

In vitro responsiveness of the horse anterior pituitary (AP) gonadotropes to single and multiple GnRH challenges was examined. The pituitaries were collected from reproductively sound mares in estrus (n = 5) and diestrus (n = 5). Uniform 0.5 mm AP slices were subdivided using a 3 mm biopsy punch and then bisected for use in the perifusion chamber. Four bisected sections per chamber were perifused at 0.5 ml/min at 37 C for 560 min in Medium 199 saturated with 95% 0(2)/5% CO2. Ten minute fractions were collected after an initial 2 hr equilibration period. Four different treatment regimes of GnRH (10(-10) M) were evaluated: (A) three consecutive 10 min GnRH pulses separated by 80 and 100 min, respectively; (B) a single 120 min GnRH infusion; (C) a 10 min GnRH pulse followed 80 min later by a 120 min GnRH infusion and (D) two 10 min GnRH pulses separated by 60 min followed 80 min later by a 120 min GnRH infusion. Estimated total pituitary LH content was higher in estrous than diestrus mares (p less than 0.05). The total amount of LH released in response to GnRH tended to be greater in estrus than diestrus (p less than 0.1), whereas the percentage of LH released in estrus and diestrus was similar. An increase in the area under the LH response curve was noted with each successive 10 min pulse of GnRH during both estrus and diestrus (p less than 0.05), demonstrating a self-priming effect of GnRH. In addition, a significant increase in the peak LH amplitude (p less than 0.05) and the slope to peak amplitude (p less than 0.05) were observed for the 120 min GnRH pulse in regime C and D indicating that prior exposure to short-term pulses of GnRH increased the acute LH secretory response. These results suggest that in the cycling mare (1) the responsiveness of the pituitary (amount of LH released as percent of total LH) is similar in both estrus and diestrus, however, the magnitude of the LH response (total microgram amount of LH released) differs with the stage of the estrous cycle, being highest in estrus, and appears to be related, in part, to pituitary LH content and (2) GnRH self-priming occurs independently of the stage of the estrous cycle. Furthermore, we have demonstrated that the pulsatile mode of GnRH can act directly on the anterior pituitary to dictate the pulsatile release pattern of LH in the cycling mare.

Animals↗

PubMed1991

Circulating antagonist of luteinizing hormone in association with infertility in stallions.

Using a LH radioligand receptor assay (RRA) previously validated for use in serum and an equine monoclonal RIA, we have distinguished a subset of subfertile stallions with an elevated RRA/RIA ratio. After purification of the active moiety by anion exchange chromatography and immunoprecipitation with the equine LH (eLH) monoclonal antibody, RRA activity remained in the supernatant. This activity was also recognized by a polyclonal LH antibody (GDN 15) with wide cross-species recognition. This active fraction was further purified by gel filtration chromatography and shown to displace labeled eLH in a dose-dependent fashion in the RRA with an inhibition slope of 2.8 compared with a slope of 1.1 for native eLH. This fraction also inhibited the LH-stimulated steroidogenesis of Leydig cells in vitro in a dose-dependent fashion, but had no effect on basal (minus LH) steroid production. Polyacrylamide gel electrophoresis and electroelution of this material demonstrated RRA activity in a fraction with a mol wt between 45-66 kDa. We conclude that this substance 1) competitively inhibited binding of eLH and hCG to the LH receptor, 2) antagonized LH-stimulated steroidogenesis in vitro, and 3) may represent a LH isoform found in association with infertility in these animals.

Animals↗