Increase In LH Concentrations Research Articles

Validation of a simple, sensitive enzyme immunoassay (EIA) for the determination of caprine plasma LH

The study describes the development and validation of a simple and highly sensitive enzyme immunoassay (EIA) for the determination of goat plasma LH, utilizing the biotin–streptavidin peroxidase amplification system in a competitive-binding assay. Micro-titer plates were coated with goat anti-rabbit globulin as the second antibody and biotin was coupled to oLH and used as a bridge between streptavidin peroxidase and immobilized oLH beta antisera. A simple 4-step procedure was used at room temperature for the sample analysis: (1) overnight incubation of LH standards and plasma samples with the LH antibody in a 96-well micro-titer plate, pre-coated with a second antibody; (2) incubation in a biotinylated–LH conjugate for 30 min; (3) incubation with streptavidin peroxidase for a further 30 min and (4) lastly incubation in tetramethyl benzidine substrate for 40 min to develop colour. A two-dimensional titer determination test proved the antibody titer of 1:100,00,000 and the biotinylated–LH conjugate titer of 1:4000 to be the most suitable. As the absolute binding sensitivity of different concentrations of oLH in 80 μl plasma was similar to that observed in buffer standards, all assays were conducted using 80 μl of the unknown plasma samples. A standard curve was obtained in the range of 25–12,800 pg LH/well in 80 μl hormone-free plasma. The sensitivity of the EIA procedure was 25 pg/well LH, which corresponds to 0.31 ng/ml plasma. In a parallelism test, the relative percentage binding curve for serially diluted goat plasma samples containing high levels of endogenous LH and for the oLH standard ran parallel to each other, thereby confirming the actual LH estimation in goat plasma. The classical patterns of plasma LH during the estrous cycle and the gradual increases in LH concentrations after gonadotrophin releasing hormone (GnRH) administration provided biological validation of the assay for the determination of plasma LH in caprine species. This EIA technique provides a simple and sensitive method for routine analysis of goat plasma LH.

Disruption of the periovulatory LH surge by a transient increase in circulating 17β-estradiol at the time of ovulation in mares

The mechanism for a reported temporal association between ovulation and a transient disruption in the periovulatory increase in LH concentrations was studied in nine mares treated with human chorionic gonadotropin when the preovulatory follicle was ≥32 mm. Examinations for ovulation detection and blood collection were done at 2-h intervals and the results were retrospectively centralized to ovulation (Hour 0). Concentrations of LH began to increase ( P < 0.03) rapidly at Hour −18, decreased ( P < 0.04) between Hours 0 and 6, and again increased ( P < 0.0001) after Hour 12. A progressive decrease ( P < 0.0001) in estradiol between Hours −30 and 24 was interrupted temporarily by an increase ( P < 0.01) between Hours −2 and 0 and a decrease ( P < 0.007) between Hours 0 and 2. Results indicated that a disruption and depression in the periovulatory LH surge occurred at the time of detected ovulation in mares and was temporally associated with a transient increase in estradiol during an overall progressive decline. The disruption in LH concentrations is attributable to the estradiol increase, based on a reported negative effect of estradiol on LH. The source of the circulating estradiol was likely from the discharge of estradiol-laden follicular fluid into the abdomen during ovulation and rapid absorption of estradiol into the circulation.

Fasting-induced suppression of LH secretion does not require activation of ATP-sensitive potassium channels

Reproductive hormone secretions are inhibited by fasting and restored by feeding. Metabolic signals mediating these effects include fluctuations in serum glucose, insulin, and leptin. Because ATP-sensitive potassium (K(ATP)) channels mediate glucose sensing and many actions of insulin and leptin in neurons, we assessed their role in suppressing LH secretion during food restriction. Vehicle or a K(ATP) channel blocker, tolbutamide, was infused into the lateral cerebroventricle in ovariectomized mice that were either fed or fasted for 48 h. Tolbutamide infusion resulted in a twofold increase in LH concentrations in both fed and fasted mice compared with both fed and fasted vehicle-treated mice. However, tolbutamide did not reverse the suppression of LH in the majority of fasted animals. In sulfonylurea (SUR)1-null mutant (SUR1(-/-)) mice, which are deficient in K(ATP) channels, and their wild-type (WT) littermates, a 48-h fast was found to reduce serum LH concentrations in both WT and SUR(-/-) mice. The present study demonstrates that 1) blockade of K(ATP) channels elevates LH secretion regardless of energy balance and 2) acute fasting suppresses LH secretion in both SUR1(-/-) and WT mice. These findings support the hypothesis that K(ATP) channels are linked to the regulation of gonadotropin-releasing hormone (GnRH) release but are not obligatory for mediating the effects of fasting on GnRH/LH secretion. Thus it is unlikely that the modulation of K(ATP) channels either as part of the classical glucose-sensing mechanism or as a component of insulin or leptin signaling plays a major role in the suppression of GnRH and LH secretion during food restriction.

Relationship Among Follicular Growth, Oestrus, Time of Ovulation, Endogenous Estradiol 17β and Luteinizing Hormone in Bos Indicus Cows After a Synchronization Program

To determine the pattern of follicular growth during oestrus and the relationship with estradiol and luteinizing hormone in ovulating and non-ovulating cows, three groups of (n = 10), thirty cyclic, Bos indicus cows were synchronized with CIDR, consecutively at 9-day intervals. Twenty-four hours after implant withdrawal, all cows synchronized in the same group with other cows displaying estrous behaviour after implant withdrawal were subjected to an intensive period of ultrasonographic observations (every 6 h for 120 h). Blood samples were taken to evaluate LH surge and 17-beta estradiol. No differences were observed in follicular growth, ovulatory diameter and growth average in the three groups of synchronized cows. Cows ovulating (CO) had a better growth average in comparison with the group of cows not ovulating (CNO) (1.4 +/- 0.7 mm vs 0.7 +/- 0.5 mm, p < 0.06). The average time from estradiol release to LH surge was 39.3 +/- 24.6 h. Differences were also observed between CO and CNO with respect to both the first concentration (27.7 +/- 5.2 vs 58.6 +/- 31.9, p < 0.004) and last concentration (79.3 +/- 23.3 vs 99.2 +/- 27.3, p < 0.05) of estradiol above 5 pg/ml. The average time from overt signs of oestrus to LH release was 8.4 +/- 7.7 h. In the CNO, the increase in LH concentration was never above two SD from the basal average. In conclusion, there is a wide variability in follicular growth and ovulatory diameter between CO and CNO, which can affect the intervals of LH release, estradiol peak and ovulation. Yet, LH surge might be a good marker for timing ovulation in Zebu cows.

The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season

Application of the ram effect during the breeding season has been previously disregarded because the ewe reproductive axis is powerfully inhibited by luteal phase progesterone concentrations. However, anovulatory ewes treated with exogenous progestagens respond to ram introduction with an increase in LH concentrations. We therefore tested whether cyclic ewes would respond to ram introduction with an increase in pulsatile LH secretion at all stages of the estrous cycle. We did two experiments using genotypes native to temperate or Mediterranean regions. In Experiment 1 (UK), 12 randomly cycling, North of England Mule ewes were introduced to rams midway through a frequent blood-sampling regime. Ewes in the early (EL; n = 6) and late luteal (LL; n = 6) phase responded to ram introduction with an increase in LH pulse frequency and mean and basal concentrations of LH (at least P < 0.05). In Experiment 2 (Australia), the cycles of 32 Merino ewes were synchronised using intravaginal progestagen pessaries. Pessary insertion was staggered to produce eight ewes at each stage of the estrous cycle: follicular (F), early luteal (EL), mid-luteal (ML) and late luteal (LL). In all stages of the cycle, ewes responded to ram introduction with an increase in LH pulse frequency ( P < 0.01); EL, ML and LL ewes also had an increase in mean LH concentration ( P < 0.05). In conclusion, ram introduction to cyclic ewes stimulated an increase in pulsatile LH secretion, independent of ewe genotype or stage of the estrous cycle.

Endocrine regulations of reproductive seasonality, follicular development and incubation in Magang geese

This study investigated the regulatory mechanisms of seasonal breeding, developments of ovarian follicles and incubation in Magang geese, a short day breeding bird. Throughout the year, plasma PRL concentrations increased in non-breeding season in spring and summer (from April to early August), and remained low in the rest of the year, while LH concentrations peaked in August and September and remained low in non-breeding season (March to June). Lengthening photoperiod increased PRL and decreased LH secretions, which inhibited follicular development, terminated lay and induced moulting, while shortening photoperiod decreased PRL and increased LH secretion and reinitiated lay. Long photoperiod stimulated PRL secretion occurred with increased gene expressions of PRL in the pituitary gland and VIP in the hypothalamus, but inhibition of LH secretion was without decreases in gene expressions of LH β subunit and GnRH. Under breeding conditions, terminating incubation decreased PRL but increased LH concentrations and resumed lay in 24 days following recruitment of about 10 large white follicles into hierarchical development. Plasma concentrations of progesterone and inhibin peaked at peak lay, whereas LH concentrations exhibited a bi-phasic pattern with troughs at peak lay and incubation when PRL concentrations were high. Ninety percent geese exhibited incubation behaviour after laying one clutch of approximately eight eggs in approximately 30 days. In conclusion the seasonal reproductive activities in Magang geese is directly inhibited by long photoperiod and directly stimulated by short photoperiod via PRL and LH secretions, whose interplays also cause occurrences of four to five lay and incubation cycles in the breeding season.

The origins and sequelae of abnormal neuroendocrine function in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common clinical disorder characterized by ovulatory dysfunction and hyperandrogenaemia. A neuroendocrine hallmark of PCOS is persistently rapid LH (GnRH) pulsatility, which favours pituitary synthesis of LH over that of FSH and contributes to the increased LH concentrations and LH : FSH ratios typical of PCOS. Inadequate FSH levels contribute to impaired follicular development, whereas elevated LH levels augment ovarian androgen production. Whereas luteal phase elevations in progesterone normally slow GnRH pulse frequency, women with PCOS do not experience normal progesterone-mediated slowing, due in part to impaired hypothalamic progesterone sensitivity. This reduction in hypothalamic progesterone sensitivity appears to be mediated by elevated androgens because sensitivity can be restored with the androgen receptor blocker flutamide. The ovulatory and hormonal abnormalities associated with PCOS generally present during puberty, typically associated with hyperandrogenaemia. Along with elevated LH concentration and pulsatility, some girls with hyperandrogenaemia have impaired hypothalamic progesterone sensitivity similar to that seen in adult women with PCOS. We propose that peripubertal hyperandrogenaemia may lead to persistently rapid GnRH pulse frequency via impaired hypothalamic feedback inhibition. The subsequent abnormalities in gonadotropin secretion, androgen production and ovulatory function may support progression towards the adult PCOS phenotype.

Development and Validation of a Simple, Sensitive, Second Antibody Format Enzyme Immunoassay (EIA) for LH Determination in Mithun (Bos Frontalis) Plasma

The objective of this study was to develop and validate a simple and highly sensitive enzyme immunoassay (EIA) for LH determination in mithun plasma on microtitreplates using the biotin‐streptavidin amplification system and the second antibody coating technique. Biotin was coupled to LH and used to bridge between streptavidin‐peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 20 µL mithun plasma. The LH standards ranging from 6.25 pg/well/20 µL to 400 pg/well/20 µL were prepared in hormone free plasma collected from a mithun on day 3 post calving. The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/mL plasma; the 50 percent relative binding sensitivity was seen at 100 pg/well/20 µL. Plasma volumes for the EIA viz. 10 and 20 µL did not influence the shape of standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma LH with bovine LH standards. It showed good parallelism with the bovine standard curve. For the biological validation of the assay, 3 mithuns were used. These were administered 10 µg i.v., with a synthetic analogue of GnRH (Buserelin‐Acetate, Intervet, India) and blood samples were collected at 15 min intervals using indwelling jugular catheter beginning 1 h prior to GnRH injection till 8 h post injection. In all animals, sharp increases in LH concentrations were recorded post GnRH administration, which confirms the biological validation of the EIA. In conclusion, the EIA developed for LH determination in mithun blood plasma is sufficiently reliable, economical, and sensitive enough to estimate LH in all physiological variations in mithun.

Effect of plane of nutrition on seasonality of reproduction in Spanish Payoya goats

The aim of this study was to determine if there is a seasonal pattern of sexual activity in female Payoya goats and if this seasonality could be modulated by nutrition. During the experimental period of 20 months, 43 non-pregnant adults goats were penned under natural photoperiod at latitude 37°15′N. At the onset of the experiment, the animals were allocated to three experimental groups differing in the level of nutrition and whether the animals were entire or ovariectomized does. The high nutrition group (H, n = 16 entire does) receiving 1.5 times maintenance requirements. The low nutrition group (L, n = 16 entire does) and an ovariectomized and oestradiol treated group (OVX, n = 11 ovariectomized does) received a diet supporting their maintenance requirements. The groups were balanced for live weight (LW) and body condition score (BCS) at the beginning of the study. In entire goats, oestrus was tested daily using aproned males, ovulation rate was assessed by laparoscopy 7 days after identification of oestrus and plasma samples were obtained twice per week for progesterone assay. OVX goats were isolated from the other groups and bucks, plasma samples were assayed twice per week for LH and there were four intensive sampling periods during the year to determine LH pulsatility. LW and BCS were recorded for all animals once a week. A clear circannual cycle in live weight change was observed in all experimental groups, being relatively stable or slightly decreasing in summer and autumn and increasing during winter and spring. The effect of exposure to high (H) rather than low (L) nutrition was to cause earlier onset of ovarian activity (5 versus 17 August; P < 0.05), and expression of oestrous (16 August versus 2 September; P < 0.01) and later cessation of reproductive activity (ovulation 11 February versus 17 January; P < 0.01). Consequently, seasonal anoestrus was 32 days shorter in does on the higher plane of nutrition ( P < 0.01). The seasonality of reproductive activity was confirmed in the OVX does, with reduced LH concentrations during spring and summer, and increased LH concentrations in autumn and winter. There was no effect of nutrition on ovulation rate. These results demonstrate that the female Payoya goat exhibits marked reproductive seasonality which is modulated by nutrition but possibly not ovulation rate.

Puberty in South American Bos indicus (Zebu) cattle

Puberty in Zebu heifers follows a pattern characterized by a decrease in the steroid feedback mechanism and an increase in LH concentration, which result in the first ovulation followed by a short estrous cycle and the onset of normal cycles thereafter. These events are similar to those observed in Bos taurus cattle but occur at a later age. The late onset of puberty is both genetic and environmental in origin and is reflected by the age at first calving that can be at 40 months of age or older in these animals. Age at puberty in Zebu heifers has been shown to have a high heritability. Consequently, selecting precocious heifers may be an effective means of reducing age at puberty in these animals and this approach is being adopted in commercial practice. Genetic selection is not the sole solution to the problem because environmental improvements are necessary, particularly in terms of improved nutrition. South American Zebu cattle are usually subject to sub-optimum nutritional and management conditions and, hence, exhibit late onset of puberty. Hybrids of Zebu and Bos taurus cattle exhibit heterosis in respect of the age of puberty with earlier onset than expected in crossbred animals. Recently, purebred South American Zebu cattle have been shown to have Bos taurus genes, indicating that there have been previous attempts to improve their productivity using this approach. It was concluded that the age at first calving in South American Zebu cattle can be reduced by exposing well-fed, yearling heifers to bulls and selecting, over several generations, those animals that become pregnant at an early age.

Puberty in South American Bos indicus (Zebu) cattle

Puberty in Zebu heifers follows a pattern characterized by a decrease in the steroid feedback mechanism and an increase in LH concentration, which result in the first ovulation followed by a short estrous cycle and the onset of normal cycles thereafter. These events are similar to those observed in Bos taurus cattle but occur at a later age. The late onset of puberty is both genetic and environmental in origin and is reflected by the age at first calving that can be at 40 months of age or older in these animals. Age at puberty in Zebu heifers has been shown to have a high heritability. Consequently, selecting precocious heifers may be an effective means of reducing age at puberty in these animals and this approach is being adopted in commercial practice. Genetic selection is not the sole solution to the problem because environmental improvements are necessary, particularly in terms of improved nutrition. South American Zebu cattle are usually subject to sub-optimum nutritional and management conditions and, hence, exhibit late onset of puberty. Hybrids of Zebu and Bos taurus cattle exhibit heterosis in respect of the age of puberty with earlier onset than expected in crossbred animals. Recently, purebred South American Zebu cattle have been shown to have Bos taurus genes, indicating that there have been previous attempts to improve their productivity using this approach. It was concluded that the age at first calving in South American Zebu cattle can be reduced by exposing well-fed, yearling heifers to bulls and selecting, over several generations, those animals that become pregnant at an early age.

Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows

Haemodynamic changes are involved in the cyclic remodelling of ovarian tissue that occurs during final follicular growth, ovulation and new corpus luteum development. The aim of this study was to characterize the real-time changes in the blood flow within the follicle wall associated with the LH surge, ovulation and corpus luteum development in cows. Normally cyclic cows with a spontaneous ovulation (n = 5) or a GnRH-induced ovulation (n = 5) were examined by transrectal colour and pulsed Doppler ultrasonography to determine the area and the time-averaged maximum velocity (TAMXV) of the blood flow within the preovulatory follicle wall and the early corpus luteum. Ultrasonographic examinations began 48 h after a luteolytic injection of PGF(2alpha) analogue was given at the mid-luteal phase of the oestrous cycle. Cows with spontaneous ovulation were scanned at 6 h intervals until ovulation occurred. Cows with GnRH-induced ovulation were scanned just before GnRH injection (0 h), thereafter at 0.5, 1, 2, 6, 12, 24 h and at 24 h intervals up to day 5. Blood samples were collected at the same time points for oestradiol, LH and progesterone determinations. Cows with both spontaneous and GnRH-induced ovulation showed a clear increase in the plasma concentration of LH (LH surge) followed by ovulation 26-34 h later. In the colour Doppler image of the preovulatory follicle, the blood flow before the LH surge was detectable only in a small area in the base of the follicle. An acute increase in the blood flow velocity (TAMXV) was detected at 0.5 h after GnRH injection, synchronously with the initiation of the LH surge. At 12 h after the LH surge, the plasma concentrations of oestradiol decreased to basal concentrations. The TAMXV remained unchanged after the initial increase until ovulation, but decreased on day 2 (12-24 h after ovulation). In the early corpus luteum, the blood flow (area and TAMXV) gradually increased in parallel with the increase in corpus luteum volume and plasma progesterone concentration from day 2 to day 5, indicating active angiogenesis and normal luteal development. Collectively, the complex structural, secretory and functional changes that take place in the ovary before ovulation are closely associated with a local increase in the blood flow within the preovulatory follicle wall. The result of the present study provides the first visual information on vascular and blood flow changes associated with ovulation and early corpus luteum development in cows. This information may be essential for future studies involving pharmacological control of blood flow and alteration of ovarian function.

Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows

Haemodynamic changes are involved in the cyclic remodelling of ovarian tissue that occurs during final follicular growth, ovulation and new corpus luteum development. The aim of this study was to characterize the real-time changes in the blood flow within the follicle wall associated with the LH surge, ovulation and corpus luteum development in cows. Normally cyclic cows with a spontaneous ovulation (n = 5) or a GnRH-induced ovulation (n = 5) were examined by transrectal colour and pulsed Doppler ultrasonography to determine the area and the time-averaged maximum velocity (TAMXV) of the blood flow within the preovulatory follicle wall and the early corpus luteum. Ultrasonographic examinations began 48 h after a luteolytic injection of PGF(2alpha) analogue was given at the mid-luteal phase of the oestrous cycle. Cows with spontaneous ovulation were scanned at 6 h intervals until ovulation occurred. Cows with GnRH-induced ovulation were scanned just before GnRH injection (0 h), thereafter at 0.5, 1, 2, 6, 12, 24 h and at 24 h intervals up to day 5. Blood samples were collected at the same time points for oestradiol, LH and progesterone determinations. Cows with both spontaneous and GnRH-induced ovulation showed a clear increase in the plasma concentration of LH (LH surge) followed by ovulation 26-34 h later. In the colour Doppler image of the preovulatory follicle, the blood flow before the LH surge was detectable only in a small area in the base of the follicle. An acute increase in the blood flow velocity (TAMXV) was detected at 0.5 h after GnRH injection, synchronously with the initiation of the LH surge. At 12 h after the LH surge, the plasma concentrations of oestradiol decreased to basal concentrations. The TAMXV remained unchanged after the initial increase until ovulation, but decreased on day 2 (12-24 h after ovulation). In the early corpus luteum, the blood flow (area and TAMXV) gradually increased in parallel with the increase in corpus luteum volume and plasma progesterone concentration from day 2 to day 5, indicating active angiogenesis and normal luteal development. Collectively, the complex structural, secretory and functional changes that take place in the ovary before ovulation are closely associated with a local increase in the blood flow within the preovulatory follicle wall. The result of the present study provides the first visual information on vascular and blood flow changes associated with ovulation and early corpus luteum development in cows. This information may be essential for future studies involving pharmacological control of blood flow and alteration of ovarian function.

Castration increases pulsatile luteinizing hormone release, but fails to diminish mounting behavior in sexually experienced bulls

We tested the hypothesis that mounting and chemoinvestigatory behaviors are testosterone-dependent in bulls. Eighteen bulls were divided into three treatment groups: intact (I), castrated (C) and castrated+testosterone (T). Sexual behaviors of all bulls were tested with an unrestrained receptive female 1 week prior to and weekly for 4 weeks after castration. Mounts with intromissions, aborted mounts and flehmen responses were quantified for each test period. In addition, patterns of LH and testosterone secretion were assessed at these times. Neither mounts with intromissions nor aborted mounts were affected by treatment. In contrast, numbers of flehmen responses were lower in C bulls than in the other groups following castration. Before castration, concentrations of LH were not different among groups and LH pulse frequency was approximately one pulse per hour. Castration resulted in a 2-fold increase in mean concentrations of LH and a 6-fold increase in LH pulse frequency. Neither mean concentration of LH nor LH pulse frequency changed in I or T bulls. The data fail to support the hypothesis that mounting behavior is T-dependent, but supports the hypothesis that this steroid hormone regulates flehmen behavior in sexually experienced bulls.

Changes in Responses to GnRH on Luteinizing Hormone and Follicle Stimulating Hormone Secretion in Prepubertal Heifers.

This study was carried out to investigate changes in pituitary response to GnRH on gonadotrophin secretion in prepubertal heifers. A total of 50 prepubertal Holstein-Friesian heifers were treated with 1 μg/kg GnRH intravenously at 1, 2, 4, 6 and 8 months of age, and plasma samples were collected from the jugular vein at 0 to 360 min after GnRH treatment. Plasma concentrations of LH and FSH were measured by RIA. Increase in concentrations of LH and FSH, induced by GnRH, were observed in all heifers from 1 month of age, and the concentrations of LH and FSH at 30 min after GnRH treatment were significantly greater than those before GnRH treatment in heifers of all ages. The time from the GnRH administration to the appearance of the peak of LH and FSH rise was similar at each age and, was prolonged with age. The peak concentration and the amount of release from the pituitary of LH rise increased with age, while those of FSH had a tendency to decrease with age. These results indicate that the pituitary gland already has reactivity to GnRH in heifers by 1 month of age, that the capacity of LH release to GnRH in the pituitary gland develops with age, and that the regulatory mechanism for the secretion of LH and FSH develops differently in prepubertal heifers. This study suggests that the development of the capacity to secrete LH secretion in response to GnRH in the pituitary gland before puberty is one of the factors for deciding the time of the onset of puberty in heifers.

Inhibition of P450 Aromatase Enhances Gonadotropin Secretion in Early and Midpubertal Boys: Evidence for a Pituitary Site of Action of Endogenous E

In early pubertal boys, E concentrations are very low. We studied the role and site of action of endogenous E in the regulation of gonadotropin secretion in early and midpubertal boys by inhibiting the action of E with a potent and specific P450 aromatase inhibitor, letrozole. A total of 35 boys who were referred to us because of suspicion of delayed puberty were included in the study. The boys were in either early or midpuberty, and they composed 3 groups: 10 boys did not receive any treatment, 12 boys received T alone, and 13 boys received T and letrozole. In the untreated group during the 5-month follow-up, no changes were observed in 17β-E2, T, basal gonadotropin, or inhibin B concentrations or in the GnRH-induced gonadotropin responses. In the T-treated group during the 5-month treatment, the T concentration increased by 55% (P < 0.05), and the 17β-E2 concentration increased by 130% (P < 0.02). Concurrently, basal gonadotropin concentrations were suppressed, but the GnRH-induced gonadotropin responses and the inhibin B concentration remained unchanged. In the T- plus letrozole-treated group during the 5-month treatment, an increase in T concentration of 606% was observed (P < 0.001), but the 17β-E2 concentration remained unchanged. The changes in the 17β-E2 concentration within 5 months in the untreated and the T- plus letrozole-treated groups were different (P < 0.02), indicating significant inhibition of endogenous E synthesis during letrozole treatment. During the T plus letrozole treatment, basal gonadotropin concentration, the GnRH-induced LH response, and inhibin B concentration increased, and the GnRH-induced FSH response did not change significantly. Serum nocturnal gonadotropin pulses were determined in 5 boys treated with T and in 5 boys treated with T plus letrozole. In the T- plus letrozole-treated group, the nocturnal LH pulse amplitude increased, and the LH pulse frequency and interpulse interval remained unchanged. In conclusion, in early and midpubertal boys, suppression of the action of E by the P450 aromatase inhibitor increased LH concentration, LH pulse amplitude, and the GnRH-induced LH response, which indicates that in boys during early and midpuberty, endogenous E regulates LH secretion at the site of the pituitary.

Inhibition of P450 aromatase enhances gonadotropin secretion in early and midpubertal boys: evidence for a pituitary site of action of endogenous E.

In early pubertal boys, E concentrations are very low. We studied the role and site of action of endogenous E in the regulation of gonadotropin secretion in early and midpubertal boys by inhibiting the action of E with a potent and specific P450 aromatase inhibitor, letrozole. A total of 35 boys who were referred to us because of suspicion of delayed puberty were included in the study. The boys were in either early or midpuberty, and they composed 3 groups: 10 boys did not receive any treatment, 12 boys received T alone, and 13 boys received T and letrozole. In the untreated group during the 5-month follow-up, no changes were observed in 17beta-E2, T, basal gonadotropin, or inhibin B concentrations or in the GnRH-induced gonadotropin responses. In the T-treated group during the 5-month treatment, the T concentration increased by 55% (P < 0.05), and the 17beta-E2 concentration increased by 130% (P < 0.02). Concurrently, basal gonadotropin concentrations were suppressed, but the GnRH-induced gonadotropin responses and the inhibin B concentration remained unchanged. In the T- plus letrozole-treated group during the 5-month treatment, an increase in T concentration of 606% was observed (P < 0.001), but the 17beta-E2 concentration remained unchanged. The changes in the 17beta-E2 concentration within 5 months in the untreated and the T- plus letrozole-treated groups were different (P < 0.02), indicating significant inhibition of endogenous E synthesis during letrozole treatment. During the T plus letrozole treatment, basal gonadotropin concentration, the GnRH-induced LH response, and inhibin B concentration increased, and the GnRH-induced FSH response did not change significantly. Serum nocturnal gonadotropin pulses were determined in 5 boys treated with T and in 5 boys treated with T plus letrozole. In the T- plus letrozole-treated group, the nocturnal LH pulse amplitude increased, and the LH pulse frequency and interpulse interval remained unchanged. In conclusion, in early and midpubertal boys, suppression of the action of E by the P450 aromatase inhibitor increased LH concentration, LH pulse amplitude, and the GnRH-induced LH response, which indicates that in boys during early and midpuberty, endogenous E regulates LH secretion at the site of the pituitary.

Response of estradiol and inhibin to experimentally reduced luteinizing hormone during follicle deviation in mares.

The increase in LH concentrations at the time of the decrease in FSH concentrations during follicle deviation in mares was studied to determine the role of LH in the production of estradiol and immunoreactive inhibin (ir-inhibin). Ten days after ovulation, all follicles > or =6 mm were ablated, prostaglandin F(2 alpha) was given, and either 0 mg (control group, n = 15) or 100 mg of progesterone in safflower oil (treated group, n = 16) was given daily for 14 days, encompassing the day of diameter deviation. The follicular and hormonal data were normalized to the expected day of the beginning of diameter deviation when the largest follicle first reached > or =20 mm (Day 0). The experimentally induced decrease in LH concentrations during follicle deviation beginning on Day -4 delayed and stunted the increase in circulating concentrations of ir-inhibin and estradiol beginning on Days -3 and -1, respectively, but did not alter the predeviation FSH surge and the initiation of diameter deviation between the two largest follicles. Combined for both groups, the interval to the expected day of deviation was 16.6 days after ovulation when the largest follicle was a mean of 21.6 mm. After deviation, the largest follicle started to regress in the treated group beginning on Day 1 and was associated with decreased concentrations of ir-inhibin and estradiol, and increased concentrations of FSH. The negative influence of the dominant follicle on the postdeviation decrease in FSH observed in the control group was alleviated and concentrations resurged in the treated group. Apparently this is the first in vivo evidence that the increase in LH that precedes follicle deviation has a positive effect in supporting the production of inhibin during diameter deviation. It was concluded that the increase in LH concentrations before diameter deviation played a role in the production of estradiol and inhibin by the largest follicle during deviation.

Effect of LH on circulating oestradiol and follicular fluid factor concentrations during follicle deviation in cattle

Progesterone was used to reduce LH concentrations starting at the time when the largest follicle was > or = 5.7 mm in diameter or well before the expected start of follicle deviation (largest follicle > or = 8.5 mm in diameter). Plasma concentrations of LH, FSH and oestradiol were determined at 4 h intervals in control and progesterone-treated heifers (n = 8 per group). Concentrations of LH were lower (P < 0.05) in the progesterone-treated group, reflecting an absence of the transient increase in LH concentrations that encompasses follicle deviation. An increase in oestradiol and a continued decrease in FSH occurred at the start of follicle deviation in the control cows but not in the treated heifers. In a second experiment, follicular fluid of the two largest follicles of control and progesterone-treated heifers was sampled at the expected start of deviation (n = 8--10 per group). The concentrations of oestradiol, but not androstenedione and free insulin-like growth factor I (IGF-I), in follicular fluid were higher (P < 0.001) in the largest follicle than in the second largest follicle. Progesterone treatment reduced (P < or = 0.02) the concentrations of all three factors in follicular fluid and increased (P < 0.05) the concentrations of insulin-like growth factor binding protein 2 (IGFBP-2). These results confirm that oestradiol contributes to the continued decrease in FSH concentrations after the start of follicle deviation. Prevention of the transient LH increase, the oestradiol increase and the continued FSH decrease did not significantly alter the mean time or follicle diameters characteristic of expected follicle deviation. However, in some treated individuals (three of eight), the observed follicle deviation was delayed. In addition, these results indicate that the secretion of oestradiol into the circulation and the increase in oestradiol and IGF-I and decrease in IGFBP-2 concentrations in the follicular fluid at the start of deviation are functions of the transient increase in LH concentrations that encompasses follicle deviation.

Effect of LH on circulating oestradiol and follicular fluid factor concentrations during follicle deviation in cattle

Progesterone was used to reduce LH concentrations starting at the time when the largest follicle was > or = 5.7 mm in diameter or well before the expected start of follicle deviation (largest follicle > or = 8.5 mm in diameter). Plasma concentrations of LH, FSH and oestradiol were determined at 4 h intervals in control and progesterone-treated heifers (n = 8 per group). Concentrations of LH were lower (P < 0.05) in the progesterone-treated group, reflecting an absence of the transient increase in LH concentrations that encompasses follicle deviation. An increase in oestradiol and a continued decrease in FSH occurred at the start of follicle deviation in the control cows but not in the treated heifers. In a second experiment, follicular fluid of the two largest follicles of control and progesterone-treated heifers was sampled at the expected start of deviation (n = 8--10 per group). The concentrations of oestradiol, but not androstenedione and free insulin-like growth factor I (IGF-I), in follicular fluid were higher (P < 0.001) in the largest follicle than in the second largest follicle. Progesterone treatment reduced (P < or = 0.02) the concentrations of all three factors in follicular fluid and increased (P < 0.05) the concentrations of insulin-like growth factor binding protein 2 (IGFBP-2). These results confirm that oestradiol contributes to the continued decrease in FSH concentrations after the start of follicle deviation. Prevention of the transient LH increase, the oestradiol increase and the continued FSH decrease did not significantly alter the mean time or follicle diameters characteristic of expected follicle deviation. However, in some treated individuals (three of eight), the observed follicle deviation was delayed. In addition, these results indicate that the secretion of oestradiol into the circulation and the increase in oestradiol and IGF-I and decrease in IGFBP-2 concentrations in the follicular fluid at the start of deviation are functions of the transient increase in LH concentrations that encompasses follicle deviation.