LH Secretion Research Articles

How does nutrition influence luteal function and early embryo survival

The pre-ovulatory LH surge triggers luteinisation of follicle tissue, but subsequent development of corpora lutea to full size is independent of LH up to around day 12 of pregnancy. Thereafter, severe (pharmacological) inhibition of LH secretion for 3 to 5 days will result in luteal failure and loss of pregnancy. It is unlikely that nutritional circumstances will have a similar effect, although scenarios with severe undernutrition have hardly been studied during early pregnancy. Milder levels of pre- and postmating undernutrition (around maintenance requirements), do affect luteal tissue development, but whether this is related to variation in LH is not clear as studies are equivocal, and there are indications that other nutrition related factors, like IGF-1 and insulin, may mediate these effects. A high plane of nutrition seems to increase progesterone secretion by the ovaries, even though systemic progesterone is reduced at the same time. Since there is direct transfer of progesterone from ovarian veins to the uterus, this may explain why a high plane of nutrition may actually benefit embryo survival and pregnancy, although very early during luteal formation (first days after mating), secretion by the ovaries may be overridden by systemic clearance of progesterone on a high feed level. Direct measurement of progesterone secretion by the ovaries is poorly understood as is the transfer of progesterone to the uterus and effects of specific nutrition related mediators such as IGF-1 and insulin on these processes.

Leptin actions on the reproductive neuroendocrine axis in sheep

There is a growing literature on the role of leptin in appetite and neuroendocrine regulation in domestic ruminants. Circulating leptin concentration is higher in fat than in thin sheep, is reduced by chronic underfeeding and is higher in sheep subjected to long-day rather than short-day photoperiods. Leptin is reduced acutely by fasting and increases after meals so that there are long- and short-term components to the systemic leptin signal. Nutritional stimulation of reproductive neuroendocrine output is associated with increased circulating concentrations of leptin; peripheral leptin administration restores LH secretion in fasted sheep, and leptin is permissive (although not a trigger) for puberty. Intracerebroventricular (i.c.v.) pharmacological leptin infusion stimulates LH in underfed but not in well-fed sheep, and reduces food intake in well-fed sheep. A single i.c.v. pharmacological injection or physiological infusion of leptin stimulates LH in well-fed sheep, with or without a concomitant decrease in appetite. Furthermore, these appetite and LH responses are differentially affected by photoperiod, indicating that different neuronal pathways may mediate the two responses. Hypothalamic leptin receptors co-localize with orexigenic and anorexigenic neurones, some of which contact GnRH cells, but the confluence of leptin signalling with photoperiod (melatonin) signalling remains unresolved. Photoperiod-entrained sheep provide potential models of altered central leptin sensitivity, in which downstream mechanisms regulating appetite and GnRH may be dissociated.

Intravaginal application of Ovugel, a productcontaining the GnRH agonist triptorelin acetate (TA), stimulates LH secretion in gilts

Intravaginal application of Ovugel, a productcontaining the GnRH agonist triptorelin acetate (TA), stimulates LH secretion in gilts

Gonadotrophic control of follicle growth in the ewe

Preovulatory follicle growth in the ewe is dependent on FSH although no precise relationship appears to exist between plasma concentrations of FSH and the number of preovulatory follicles which develop or ovulation rate. This may be related to a hitherto unrecognized influence of pulsatile LH on the growth of large follicles. Preovulatory follicle growth is dependent on the presence of basal amounts of LH, but pulsatile LH, while being essential to supply an increase in androgen substrate to the granulosa cells of the follicle, may also play a role in reducing the responsiveness of many large follicles to FSH, in particular during the preovulatory phase when plasma concentrations of FSH are reduced. Thus selection of the preovulatory follicle(s) may involve a previously unrecognized interaction between FSH and pulsatile LH secretion in which pulses of LH act in a negative rather than positive manner.

The role of leptin in nutritional status and reproductive function

Infertility associated with suboptimal nutrition is a major concern among livestock producers. Undernourished prepubertal animals will not enter puberty until they are well fed; similarly, adult, normally cyclic females will stop cycling when faced with extreme undernutrition. Work in our laboratory has focused on how body fat (or adiposity) of an animal can communicate to the brain and regulate reproductive competence. In 1994, the discovery in rodents of the obese (ob) gene product leptin, secreted as a hormone from adipocytes, provided a unique opportunity to understand and hence regulate whole body compositional changes. There is now evidence that similar mechanisms are functioning in livestock species in which food intake, body composition, and reproductive performance are of considerable economic importance. Leptin has been reported to be a potent regulator of food intake and reproduction in rodents. There is evidence indicating that at least some of the effects of leptin occur through receptor-mediated regulation of the hypothalamic protein neuropeptide Y (NPY). NPY is a potent stimulator of food intake, is present at high concentrations in feed-restricted cattle and ewes, and is an inhibitor of LH secretion in these livestock species. In our investigations in sheep, we have cloned a partial cDNA corresponding to the ovine long-form leptin receptor, presumably the only fully active form, and have localized the long-form leptin receptor in the ventromedial and arcuate nuclei of the hypothalamus. Leptin receptor mRNA expression was colocalized with NPY mRNA-containing cell bodies in those regions. We have also determined that hypothalamic leptin receptor expression is greater in feed-restricted ewes than in well-fed ewes. These observations provide a foundation for future investigations into the nutritional modulators of reproduction in livestock.

Follicle growth, corpus luteum function and their effects on embryo development in postpartum dairy cows

Absent or irregular ovarian cycles in lactating dairy cows are caused by failure to ovulate the dominant follicle at the appropriate time. The follicle then either regresses or develops into a cyst. This process can be triggered by a variety of metabolic and disease factors that act at the hypothalamus and pituitary gland to inhibit pulsatile LH secretion and the LH surge, and at the ovary to reduce follicular growth and oestradiol production. Cows of poor energy status have low circulating concentrations of insulin-like growth factor I (IGF-I). Predisposing factors include calving difficulties, inappropriate diet, reduced intake of dry matter and a high rate of body condition score loss. Various stressors predispose the follicle to cyst development by inhibiting the LH surge and ovulation; these include common infections, such as mastitis. Even when ovulation does occur, poor follicular development may result in production of an inadequate corpus luteum. The timing of the increase in progesterone in the early luteal phase (days 4–5) appears to be a key determinant of fertility, probably because it alters the secretory activity of the reproductive tract, thus influencing embryonic growth and interferon-τ production. A period of negative energy balance after calving can reduce fertility even though metabolic parameters have apparently improved at the time of service.

Dopaminergic control of LH secretion by the Al5 nucleus in anoestrous ewes

Dopaminergic control of LH secretion by the Al5 nucleus in anoestrous ewes

Stress and the control of LH secretion in the ewe

Stress influences the activity of the reproductive system at several sites. One of the most significant effects is at level of the GnRH secretory system to reduce GnRH pulsatility and thus LH pulsatility. This in turn reduces the oestradiol signal that stimulates the GnRH–LH surge in the follicular phase. Three sequential phases have been identified in the induction of the GnRH–LH surge by oestradiol: (i) activation, (ii) transmission and (iii) surge secretion. There is evidence that administration of endotoxin prevents activation but not transmission, hypoglycaemia blocks both activation and transmission, whereas truck transport is effective during the late, but not early, transmission phase. Opioids mediate the suppressive effects of hypoglycaemia on both LH pulsatility and the delayed onset of the LH surge in ewes. The exact neurocircuitry used in sheep is yet to be identified but many of the connections that are proposed as important in rats are present in sheep. Corticotrophin-releasing hormone (CRH) neurones in the paraventricular nucleus that project axons to the median eminence probably do not directly inhibit GnRH, but cither afferent or parallel central pathways are involved. New members of the CRH peptide and receptor families have been identified, but roles in the control of reproduction have yet to be determined.

Tetrapleura tetraptera Extract Inhibited Luteinizing Hormone and Estrogen Secretion in Clomiphene Citrate Treated Female Wistar Albino Rats

Tetrapleura tetraptera stem bark has been reported to cause inhibition of luteinizing hormone release in cultured rat pituitary cells. Hence, we investigated the effects of Tetraptera tetrapleura pod extract on follicle stimulating hormone, luteinizing hormone, cortisol, progesterone and estrogen. Thirty non-pregnant female wistar albino rats were divided into group A - D. Group A rats were used as Control. Group B rats were administered 1 mg/kg/day of clomiphene citrate orally. Group C rats were administered 200 mg/kg of extract only, whereas group D rats were administered 1 mg/kg /day of Clomiphene citrate plus 200 mg/kg of extract. At the end of 14 days experiment, group A, B and D were found in proestrus phase and group C in diestrus phase. FSH and cortisol levels remained unchanged. Group C and D rats produced significant reduction (P < 0.05) in LH and estrogen levels in prolonged proestrus and normal diestrus respectively. Progesterone level was significantly high (P < 0.05) in the group C rats. The reduced LH level could be due to the anti-estrogenic effect of extract during proestrus when LH secretion is expected to surge. But co-administration resulted in high progesterone secretion, suggesting extract may have influenced progesterone secretion in group D rats simultaneously administered with clomiphene citrate and extract. The above findings indicated that Tetrapleura tetraptera pod extracts inhibited luteinizing hormone and estrogen even when co-administered with clomiphene citrate.

Acute Suppression of LH Secretion by Prolactin in Female Mice Is Mediated by Kisspeptin Neurons in the Arcuate Nucleus.

Hyperprolactinemia causes infertility, but the specific mechanism is unknown. It is clear that elevated prolactin levels suppress pulsatile release of GnRH from the hypothalamus, with a consequent reduction in pulsatile LH secretion from the pituitary. Only a few GnRH neurons express prolactin receptors (Prlrs), however, and thus prolactin must act indirectly in the underlying neural circuitry. Here, we have tested the hypothesis that prolactin-induced inhibition of LH secretion is mediated by kisspeptin neurons, which provide major excitatory inputs to GnRH neurons. To evaluate pulsatile LH secretion, we collected serial blood samples from diestrous mice and measured LH levels by ultrasensitive ELISA. Acute prolactin administration decreased LH pulses in wild-type mice. Kisspeptin neurons in the arcuate nucleus and in the rostral periventricular area of the third ventricle (RP3V) acutely responded to prolactin, but prolactin-induced signaling in kisspeptin neurons was up to fourfold higher in the arcuate nucleus when compared with the RP3V. Consistent with this, conditional knockout of Prlr specifically in arcuate nucleus kisspeptin neurons prevented prolactin-induced suppression of LH secretion. Our data establish that during hyperprolactinemia, suppression of pulsatile LH secretion is mediated by Prlr on arcuate kisspeptin neurons.

Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Polycystic ovary syndrome (PCOS) is a hypothalamic-pituitary-gonadal (HPG) axis disorder. PCOS symptoms most likely result from a disturbance in the complex feedback regulation system of the HPG axis, which involves gonadotrophic hormones and ovarian steroid hormones. However, the nature of this complex and interconnecting feedback regulation makes it difficult to dissect the molecular mechanisms responsible for PCOS phenotypes. Global estrogen receptor α (ERα) knockout (KO) mice exhibit a disruption of the HPG axis, resulting in hormonal dysregulation in which female ERα KO mice have elevated levels of serum estradiol (E2), testosterone, and LH. The ERα KO females are anovulatory and develop cystic hemorrhagic ovaries that are thought to be due to persistently high circulating levels of LH from the pituitary. However, the role of ERα in the pituitary is still controversial because of the varied phenotypes reported in pituitary-specific ERα KO mouse models. Therefore, we developed a mouse model where ERα is reintroduced to be exclusively expressed in the pituitary on the background of a global ERα-null (PitERtgKO) mouse. Serum E2 and LH levels were normalized in PitERtgKO females and were comparable to wild-type serum levels. However, the ovaries of PitERtgKO adult mice displayed a more overt cystic and hemorrhagic phenotype when compared with ERα KO littermates. We determined that anomalous sporadic LH secretion caused the severe ovarian phenotype of PitERtgKO females. Our observations suggest that pituitary ERα is involved in the estrogen negative feedback regulation, whereas hypothalamic ERα is necessary for the precise control of LH secretion. Uncontrolled, irregular LH secretion may be the root cause of the cystic ovarian phenotype with similarities to PCOS.-Arao, Y., Hamilton, K. J., Wu, S.-P., Tsai, M.-J., DeMayo, F. J., Korach, K. S. Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Functional Hypogonadotropic Hypogonadism in Men: Underlying Neuroendocrine Mechanisms and Natural History.

After completion of puberty a subset of men experience functional hypogonadotropic hypogonadism (FHH) secondary to excessive exercise or weight loss. This phenomenon is akin to hypothalamic amenorrhea (HA) in women, yet little is known about FHH in men. To investigate the neuroendocrine mechanisms, genetics, and natural history underlying FHH. Retrospective study in an academic medical center. Healthy postpubertal men presenting with symptoms of hypogonadism in the setting of excessive exercise (>10 hours/week) or weight loss (>10% of body weight). Healthy age-matched men served as controls. Clinical assessment, biochemical and neuroendocrine profiling, body composition, semen analysis, and genetic evaluation of genes known to cause isolated GnRH deficiency. Reproductive hormone levels, endogenous GnRH-induced LH pulse patterns, and rare genetic variants. Ten men with FHH were compared with 18 age-matched controls. Patients had significantly lower body mass index, testosterone, LH, and mean LH pulse amplitudes yet normal LH pulse frequency, serum FSH, and sperm counts. Some patients exhibited nocturnal, sleep-entrained LH pulses characteristic of early puberty, and one FHH subject showed a completely apulsatile LH secretion. After decreased exercise and weight gain, five men with men had normalized serum testosterone levels, and symptoms resolved. Rare missense variants in NSMF (n = 1) and CHD7 (n = 1) were identified in two men with FHH. FHH is a rare, reversible form of male GnRH deficiency. LH pulse patterns in male FHH are similar to those observed in women with HA. This study expands the spectrum of GnRH deficiency disorders in men.

Luteinizing hormone signaling is involved in synchronization of Leydig cell's clock and is crucial for rhythm robustness of testosterone production†.

In mammals, circadian clock regulates concentration of many reproductive hormones including testosterone. Previously, we characterized pattern of circadian transcription of core clock genes in testosterone-producing Leydig cells. Here, the potential role of luteinizing hormone receptor (LHR)-cAMP signaling in synchronization of Leydig cell's circadian clock and rhythmic testosterone production were examined. Results showed that activation of LHR-cAMP signaling in primary rat Leydig cell culture increased Star/STAR and changed expression of many clock genes (upregulated Per1/PER1, Dec1/2, and Rorb, and downregulated Bmal1 and Rev-erba/b). Inhibition of protein kinase A prevented LHR-triggered increase in transcription of Per1 and Dec1. Effect of stimulated LHR-cAMP signaling on Leydig cell's clock transcription was also confirmed in vivo, using rats treated with single hCG injection. To analyze in vivo effect of low LH-cAMP activity on rhythmical Leydig cell function, rats with experimental hypogonadotropic hypogonadism were used. Characteristics of hypogonadal rats were decreased LH and testosterone secretion without circadian fluctuation; in Leydig cells decreased arrhythmic cAMP and transcription of steroidogenic genes (Cyp11a1 and Cyp17a1) were observed, while decreased Star/STAR expression retains circadian pattern. However, expression of clock genes, despite changes in transcription levels (increased Bmal1, Per2, Cry1, Cry2, Rora, Rorb, Rev-erba/b/REV-ERBB, Dec1, Csnk1e, and decreased Npas2 and PER1) kept circadian patterns observed in control groups. Altogether, the results strengthened the hypothesis about role of LH-cAMP signaling as synchronizer of Leydig cell's clock. However, clock in Leydig cells is not sufficient to sustain rhythmicity of testosterone production in absence of rhythmic activity of LH-cAMP signaling.

Characterization of GnRH Pulse Generator Activity in Male Mice Using GCaMP Fiber Photometry.

Kisspeptin neurons located in the hypothalamic arcuate nucleus are thought to represent the GnRH pulse generator responsible for driving pulsatile LH secretion. The recent development of GCaMP6 fiber photometry technology has made it possible to perform long-term recordings of the population activity of the arcuate nucleus kisspeptin (ARNKISS) neurons in conscious-behaving mice. Using this approach, we show that ARNKISS neurons in intact male mice exhibit episodes of synchronized activity that last ∼2 minutes and have a mean inter-episode interval of 166 minutes, with a very wide range (43 to 347 minutes). Gonadectomy resulted in dramatic changes in the dynamics of ARNKISS neuron behavior with temporally distinct alterations in synchronization episode (SE) amplitude (sevenfold increase), inter-SE frequency (range, 2 to 58 minutes), and duration (up to 28 minutes), including the frequent appearance of seemingly unstable clusters of doublet and triplet SEs. The combination of photometry with repeated blood sampling revealed a perfect correlation between ARNKISS neuron population SEs and LH pulses in intact and short-term gonadectomized (GDX) mice. No differences were detected in SE frequency across 24 hours in either intact or GDX mice. These observations further support a role for ARNKISS neurons as the GnRH pulse generator and show that it operates in a stochastic manner without diurnal variation in both intact and GDX male mice. The removal of gonadal steroids has multiple time-dependent effects upon ARNKISS neuron synchronizations, indicating their critical role in shaping pulse generator behavior.

Patterns of preoptic-hypothalamic neuronal activation and LH secretion in female sheep following the introduction and withdrawal of novel males.

The neuroendocrine response of female sheep to a novel male involves neural activation in the hypothalamus. However, if males are removed, the gonadotrophic signal declines, so the neural activity is likely to change. We examined Fos-immunoreactive (IR) cells in hypothalamic tissues from seasonally anovulatory female sheep exposed to males for 2 or 6h, or for 2h followed by 4h isolation from males. Control females were killed in the absence of male exposure. Male introduction increased LH secretion in all females; male removal was associated with a reduction only in mean and basal LH concentrations. Females exposed to males for 2h had more Fos-IR cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and organum vasculosum of the lamina terminalis (OVLT) than control females. Fos-IR cells in the preoptic area (POA) were only greater than in control females after 6h exposure to a male. Removal of males decreased the number of Fos-IR cells in the ARC, VMH and OVLT, but not in the POA. Thus, hypothalamic neural activation and LH secretion in female sheep are stimulated by males and decline after male removal. However, activation in the POA persists after removal and may explain the incomplete decline in the LH response.

Mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mediates the LH-induced stimulation of progesterone synthesis in the porcine corpus luteum.

Progesterone (P4) synthesized by the corpus luteum (CL) plays a key role in the establishment and maintenance of pregnancy. The LH signal is important for luteinisation and P4 synthesis in pigs. In a previous study, we demonstrated that mitogen-activated protein kinase kinase kinase 8 (MAP3K8) regulates P4 synthesis in mouse CL, but whether the function and mechanism of MAP3K8 in the pig is similar to that in the mouse is not known. Thus, in the present study we investigated the effects of MAP3K8 on porcine CL. Abundant expression of MAP3K8 was detected in porcine CL, and, in pigs, MAP3K8 expression was higher in mature CLs (or those of the mid-luteal phase) than in regressing CLs (late luteal phase). Further functional studies in cultured porcine luteal cells showed that P4 synthesis and the expression of genes encoding the key enzymes in P4 synthesis are significantly reduced when MAP3K8 is inhibited with the MAP3K8 inhibitor Tpl2 kinase inhibitor (MAP3K8i, 10μM). After 12-24h treatment of luteal cells with 100ngmL-1 LH, MAP3K8 expression and P4 secretion were significantly upregulated. In addition, the 10μM MAP3K8 inhibitor blocked the stimulatory effect of LH on P4 synthesis and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in porcine luteal cells. The LH-induced increases in MAP3K8 phosphorylation and expression, ERK1/2 phosphorylation and P4 synthesis were all blocked when protein kinase A was inhibited by its inhibitor H89 (20 μM) in porcine luteal cells. In conclusion, MAP3K8 mediates the LH-induced stimulation of P4 synthesis through the PKA/mitogen-activated protein kinase signalling pathway in porcine CL.

Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins

The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs.The advantages of this protocol include:•The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%).•The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity.•Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.

Extra Virgin Olive Oil meningkatkan Ekpresi Reseptor Estrogen α Dan Ketebalan Endometrium Rattus Norvegicus yang Dipapar Rhodamin B

Rhodamin B is harmful to the body because it is proven can increase the oxidative stress on various reproductive chain. Rhodamin B contains compounds of chlorine (Cl), is dangerous and halogen compounds are reactive. Damage to the hypothalamus will interfere with the secretion of FSH and LH cause the follicle ripening so distracted that result in a decrease in the hormone estrogen. The hormone estrogen plays an important role in the menstrual cycle and proliferation of the endometrium. This research aimed to prove the effect of Extra Virgin Olive Oil (EVOO) with the expression of estrogen receptors α and endometrial thickness of Rattus norvegicus exposed by rhodamin B. The design used true experimental with an approach of post test only control group design. The research used white rat (Rattus norvegicus wistar strain) as the sample. The results showed that EVOO could become an alternative to prevent oxidative stress due to exposure of rhodamin B.

Somatostatin-Somatostatin Receptor 2 Signaling Mediates LH Pulse Suppression in Lactating Rats.

Follicular development and ovulation are profoundly suppressed during lactation in mammals. This suppression is suggested to be mainly due to the suckling-induced inhibition of kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and consequent inhibition of pulsatile GnRH/LH release. We examined whether central somatostatin (SST) signaling mediates the suckling-induced suppression of pulsatile LH secretion. SST has been reported to be expressed in the posterior intralaminar thalamic nucleus (PIL), where the suckling stimulus is postulated to be relayed to the hypothalamus during lactation. SST inhibitory receptors (SSTRs) are abundantly expressed in the ARC, where kisspeptin/neurokinin B/dynorphin A (KNDy) neurons are located. Histological and quantitative studies revealed that the suckling stimulus increased the number of SST-expressing cells in the PIL, and Sstr2 expression in the ARC. Furthermore, a central injection of an SSTR2 antagonist caused a significant increase in pulsatile LH release in lactating rats. Double labeling of Sstr2 and the neurokinin B gene, as a marker for ARC KNDy neurons, showed Sstr2 expression was abundantly detected in the ARC, but few KNDy neurons coexpressed Sstr2 in lactating rats. Taken together, these findings suggest the suckling-induced activation of SST-SSTR2 signaling mediates, at least in part, the suppression of pulsatile LH secretion during lactation in rats, probably via the indirect effects of SST on KNDy neurons. These results provide a new aspect on the role of central SST-SSTR signaling in understanding the mechanism underlying lactational anestrus.

4 Induction of ovulation by kisspeptin in llamas

After mating, female camelids ovulate in response to nerve growth factor (NGF) present in semen (formerly referred to as ovulation-inducing factor). The ovulatory effect appears to be induced by stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) neuronal activity and LH secretion. Recent studies have identified kisspeptin as an important mediator of GnRH secretion in several species. In the present study, we tested the hypothesis that kisspeptin is involved in the ovulatory pathway in llamas and investigated the mechanism of this effect. In Experiment 1, ovarian function in non-pregnant, non-lactating adult female llamas was synchronized by intramuscular administration of a GnRH analogue (50µg of gonadorelin acetate; Fertiline, Vetoquinol, Quebec, QC, Canada). When a growing dominant follicle ≥8mm in diameter was detected, llamas were assigned randomly to 3 treatment groups and given an intravenous dose of purified seminal NGF (1mg, single dose; n=5), kisspeptin (0.1mg kg−1 of body weight, 2 doses 1h apart; n=5), or PBS (n=4). The bioactive 10 amino acid fragment of murine kisspeptin was used. Ovulation and corpus luteum development were assessed by transrectal ultrasonography every other day from the day of treatment (Day 0) to Day 8. In Experiment 2, ovarian function among female llamas was synchronized, as in Experiment 1. When a growing dominant follicle ≥8mm in diameter was detected, llamas were given kisspeptin (0.1mg kg−1 of body weight IV, 2 doses 1h apart) beginning 2h after pretreatment with either a GnRH receptor blocker (cetrorelix acetate, 1.5mg per llama IV; Sigma, Oakville, ON, Canada; n=6) or saline (n=6). Llamas were examined 48h later by transrectal ultrasonography to detect ovulation and 8 days later to determine the presence of a corpus luteum. Chi-square tests were used to compare ovulation rates, and ANOVA for repeated measures was used to compare diameter profiles of the corpus luteum. In Experiment 1, ovulation rate did not differ between the NGF and kisspeptin groups (5/5 in each; 100%) and was greater than in the control group (0/4; 0% P<0.05). Corpus luteum diameter did not differ between llamas that ovulated in response to treatment with NGF or kisspeptin (13.2±0.8 and 14.0±1.2mm on Day 8, respectively). In Experiment 2, none of the llamas pretreated with cetrorelix ovulated in response to kisspeptin treatment (0/6; 0%), whereas all of the llamas pretreated with saline ovulated in response to kisspeptin treatment and had a corpus luteum at Day 8 (6/6; 100%; P<0.05). Results supported the hypothesis that kisspeptin induces ovulation in llamas. Because a GnRH receptor antagonist blocked ovulation, our interpretation is that the site of action of kisspeptin is upstream of the pituitary gland and involves control of GnRH release from the hypothalamus. These findings raise the possibility that kisspeptin mediates the ovulation-inducing effect of NGF. Research was supported by the Natural Sciences and Engineering Research Council of Canada.