J.F. Roser

Differential luteolytic function between the physiological breeding season, autumn transition and persistent winter cyclicity in the mare

There is a well-documented increase in luteolytic failure, resulting in spontaneously prolonged corpus luteum (SPCL) function, during estrous cycles of horses in autumn. The cause of this phenomenon may be due to seasonal alterations in PGF(2alpha) and/or in prolactin (PRL) secretion around luteolysis. To investigate this, progesterone (P4), 13, 14-dihydro, 15-keto PGF(2alpha) (PGFM) and PRL concentrations were compared between summer and autumn estrous cycles during natural luteolysis and luteolysis induced by benign uterine stimulation. A single estrous cycle from mares in June-July (n=12) was compared to multiple estrous cycles from these 12 mares plus 8 additional mares in September through December. Reproductive behavior was monitored by bringing a stallion in close proximity to the mare and ovarian events by ultrasonography. Blood was collected via jugular cannula every 6h from d 13 to 17 post-ovulation in untreated control mares (n=8 summer, n=9 autumn). In treated mares, blood collection occurred at 0, 15, 30, 45, 60, 90, 120, 180 and 240min followed by 6h intervals for a total of 5d following intrauterine saline infusion on d 7 (n=4 summer, n=11 autumn). Mares failing to return to estrus for 30d received intrauterine saline and the described intensive blood sampling protocol on d 30. Progesterone and PRL were determined on daily samples and PGFM on frequent plasma collections by RIA. Duration of ovarian luteal and follicular phases, P4 and PRL concentrations and PGFM secretion around luteolysis were compared between treatments and seasons by ANOVA. Mean P4 declined from June to December in all groups. Pulses of PGFM were detected on d 13-17 in controls and d 7-11 in saline-infused mares. Pulse patterns were not different between groups. The incidence of SPCL increased during autumn in the control group. PGFM pulses were absent on d 13-17 in mares with SPCL, but PGFM pulses could be induced in these mares by saline infusion at d 30. Autumn PGFM profiles were unchanged during spontaneous or saline-induced luteolysis compared with summer. Circulating PRL increased around natural or induced luteolysis. These results provide evidence that changes in luteal function during the autumn transition are not the result of alterations in the ability of the uterus to produce PGF(2alpha) nor due to changed CL sensitivity to PGF(2alpha). We conclude that seasonal changes in luteolytic function are caused by an alteration in the signal for PGF(2alpha) release.

Localization of insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) in equine testes.

The insulin-like growth factor-I (IGF-I) is a key regulator of reproductive functions. IGF-I actions are primarily mediated by IGF-IR. The main objective of this research was to evaluate the presence of IGF-I and IGF-I Receptor (IGF-IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF-I and IGF-IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF-I and IGF-IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti-human polyclonal antibodies against IGF-I and IGF-IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre-pubertal and pubertal stages, IGF-I immunolabelling was present in spermatogonia and Leydig cells. At post-pubertal, adult and aged stages, immunolabelling of IGF-I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF-IR was observed in spermatogonia and Leydig cells at the pre-pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post-pubertal stage. Strong immunolabelling of IGF-IR was observed in spermatogonia and Leydig cells at post-puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF-I and IGF-IR was not observed in Sertoli cells. In conclusion, IGF-I is localized in equine spermatogenic and Leydig cells, and IGF-IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF-I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.

Stimulation of Sertoli Cell Proliferation: Defining the Response Interval to an Inhibitor of Estrogen Synthesis in the Boar

Sertoli cell proliferation occurs in two major waves after birth, one neonatally and another prepubertally, each contributing to final testicular size and sperm production. However, little is known about the regulation of either wave. We have previously shown that letrozole, an inhibitor of estrogen synthesis, increases Sertoli cell number and testicular size at sexual maturity in boars. These studies were conducted to determine whether letrozole affects the first or second proliferative wave. Boars were treated with letrozole during the first wave (treatment at 1, 3, and 5 weeks), less frequently (1 week of age only, or 1 and 5 weeks), on postnatal day 1, or during the second wave (weeks 11-16). Sertoli cells were enumerated in testes and estrogen concentrations were evaluated in serum and testes. Compared with vehicle controls, letrozole reduced estrogen in boars treated at weeks 1 and 5 or 1, 3, and 5, on postnatal day 1, or prepubertally. However, Sertoli cell numbers were increased only in boars treated at 1, 3, and 5 weeks of age. Neither perinatal (1 day old) nor prepubertal letrozole treatment affected Sertoli cell numbers. Hence, Sertoli cell proliferation was sensitive to letrozole only if letrozole was administered throughout the first wave, even though estrogen synthesis was effectively inhibited at all ages. These data indicate that the neonatal but not the prepubertal window of Sertoli cell proliferation is sensitive to an inhibitor of estrogen synthesis; this suggests that these two waves are differently regulated.

A synergistic effect of insulin-like growth factor (IGF-I) on equine luteinizing hormone (eLH)-induced testosterone production from cultured Leydig cells of horses.

Localization of IGF-I and IGF-IR were observed in Leydig cells of horses using immunohistochemistry (IHC), suggesting IGF-I may play a role in equine Leydig cell steroidogenesis. Previous studies in other species have indicated that IGF-I increases basal and/or LH/hCG-induced testosterone production. The objectives of this study were to (1) test the synergistic effect of IGF-I on eLH-induced testosterone production in cultured equine Leydig cells and (2) determine if this effect is reproductive stage-dependent. Testes were collected from five pubertal (1.1±0.1 year; 1-1.5 year) and eight post-pubertal (2.88±0.35 years; 2-4 years) stallions during routine castrations at the UC Davis Veterinary Hospital. Leydig cells were isolated using validated enzymatic and mechanical procedures. Leydig cells were treated without (control) or with increasing concentrations of purified pituitary-derived eLH and/or recombinant human IGF-I (rhIGF-I) and incubated under 95% air: 5% CO(2) at 32°C for 24h. After 24h, culture media was collected and frozen at -20°C until analyzed for testosterone by a validated radioimmunoassay (RIA). In pubertal stallions, treatment with both increasing concentrations of rhIGF-I and 5ng/ml of eLH failed to demonstrate a significant difference in testosterone production compared with 5ng/ml of eLH only. However, in post-pubertal stallions, a significant increase in the concentration of testosterone in culture media was observed from Leydig cells treated with various concentrations of rhIGF-I and 1 or 5ng/ml of eLH compared with 1 or 5ng/ml of eLH only. In conclusion, IGF-I has a synergistic effect on eLH-induced testosterone production in cultured equine Leydig cells from post-pubertal but not pubertal stallions.

Recommendations for clinical GnRH challenge testing of stallions

Abstract Eight mature light-breed stallions with normal testes size, sperm output and semen quality were used to evaluate response to 3 GnRH challenge regimens in the summer in southeast Texas. Gonadotropin releasing hormone (50 μg) was administered intravenously once to each of eight stallions after three days of sexual rest (50 μg GnRH-1X). The same stallions were administered either 5μg GnRH intravenously once hourly for three injections (5 μg GnRH-3X) and 15μg GnRH intravenously once (15μg GnRH-1X) one and two weeks later. Blood samples were collected prior to and at intervals after GnRH administration. Plasma was immediately separated from blood samples and was frozen until assayed for LH, FSH, estradiol and testosterone concentrations. Percentage changes in hormone concentrations from pre-treatment values (baseline) were analyzed by paired studientst-test to detect significant rises in hormone concentrations. Group mean percentage changes in hormone concentrations were analyzed by analysis of variance to compare responses among treatments. A computerized peak-detection algorithm (PC Pulsar) was used to detect peaks in LH and testosterone concentrations following 5 μg GnRH-3X and 15 μg GnRH-1X treatment. No differences (P>0.10) were detected in percentage change from baseline concentration for LH, FSH, or testosterone at one or two hours after administration of any of the three regimens of GnRH. When more frequent sampling intervals were analyzed for 5 μg GnRH-3X or 15 μg GnRH-1X treatments, no differences were detected in percentage change from baseline concentration for any hormone at 15, 30 or 60 minutes. Thereafter, percentage changes in concentrations of LH and FSH remained increased for 5μg GnRH-3X compared to 15 μg GnRH-1X treated stallions (P 0.10) were detected between 5 μg GnRH-3X and 15 μg GnRH-1X treated stallions for changes in concentrations of estradiol throughout the experiment. For 15 μg GnRH-1X treated stallions, maximum concentrations of LH in PC Pulsar-detected peaks occurred most commonly at 15 to 30 minutes (7/8 treatment periods) after GnRH injection. Maximum concentrations of testosterone in PC Pulsar-detected peaks occurred most commonly at 60–120 min (7/8 treatment periods) after GnRH injection. A protocol of blood sampling prior to, and 15, 30, 60 and 120 minutes after, intravenous administration of small doses of GnRH would be practical for challenge testing of stallions during the breeding season. In order to reduce cost of hormone assays, we suggest assay of the pre-challenge blood sample (baseline) could include LH, FSH, testosterone and estradiol concentrations (to assess overall hypothalamic-pituitary-testicularfunction), while only LH and testosterone concentrations need be determined after GnRH administration (to assess pituitary and testicular responsiveness). Assay for LH could be done on only the 15 and 30 minute post-GnRH samples, and assay for testosterone could be done on only the 60 and 120 minute post-GnRH samples. Failure to achieve approximately a 50% increase in LH concentration by 30 minutes after GnRH administration, and/or failure to achieve approximately a 100% increase in testosterone concentration by two hours after GnRH administration, could be further pursued either by treatment with increasing dosages of GnRH, or repeated administration of GnRH at hourly intervals, as has been suggested by other workers.