LH Pulse Research Articles

Granulosa cell function and oocyte competence: Super-follicles, super-moms and super-stimulation in cattle

The review presents an overview of studies that examined the effects of follicular aging and maternal aging in the bovine model. The first of three main sections is a discussion of the developmental competence of oocytes from (1) the ovulatory follicle of 2-wave and 3-wave estrous cycles, (2) dominant follicles that develop under high or low LH pulse frequency, and (3) natural versus FSH-stimulated ovulatory follicles. The second section highlights the effects of maternal aging. Maternal aging in cattle is associated with (1) elevated circulating FSH concentrations, (2) reduced response to superstimulatory treatment, and (3) markedly decreased early embryonic development in cows >12 year of age. The third and final section on superstimulation protocols addresses the effects of the duration of FSH stimulation and withdrawal (i.e., FSH “starvation” or “coasting”) on oocyte competence. Ovarian superstimulation for 4 days altered the expression of genes related to angiogenesis, and activated oxidative stress-response genes. Extending the duration of FSH stimulation from 4 to 7 days resulted in a greater and more synchronous ovulatory response and optimal oocyte maturation. The highest rates of blastocyst development in vitro were obtained when FSH support was discontinued for 44 to 68h and granulosa cell SMAD7 mRNA was predictive of this period. Longer periods of FSH starvation resulted in a loss of oocyte competence or ovulatory capability. By extending the bovine model to the transcriptome level, new approaches and treatments may be devised to resolve subfertility in women and animals.

Neurokinin B: a new player in immune/inflammatory stress-mediated suppression of reproduction.

Both immune/inflammatory and psychosocial stressors have profound effects on suppressing reproduction. Suppressing reproduction in times of stress, such as when fighting infection, or due to other environmental factors such as overcrowding, is an important shift of survival functions for a species, especially those with longer gestation periods or parental investment in rearing of offspring (1). Among other effects on reproduction, stress acts through glucocorticoids to decrease LH pulsatile secretion (2, 3). Immune/inflammatory stress, such as that induced by lipopolysaccharide (LPS) derived from Gramnegative bacteria, suppresses the GnRH and LH surge during the early preovulatory estradiol rise in sheep (4), and this suppression is independent of prostaglandins (5). In contrast, prostaglandins are mediators of LPS-induced suppression of GnRH and LH pulsatile secretion during the luteal phase in ewes (6). Cortisol treatment mimics the suppressive effects of LPS, but blocking cortisol production does not prevent LPS-induced suppression of LH pulses (7). Until now, it has not been clear which neural pathways mediate stress-induced suppression of GnRH and LH. Neurons in the arcuate nucleus of the hypothalamus coexpress kisspeptin, neurokinin B (NKB), and dynorphin A (8). Of the peptides produced by the arcuate kisspeptin, NKB, dynorphin (KNDy) neurons, NKB appears unique in differentially modulating GnRH and LH secretion based on the steroid milieu. Both NKB and a selective neurokinin-3 receptor (NK3R) agonist have differential effects on GnRH and LH secretion, depending on the gonadal state of the subjects treated (9). For example, in female gonadal-intact, estradiol-treated rats and sheep, and pubertal rats, NKB or NK3R agonist treatment increases GnRH and LH secretion, whereas in ovariectomized rats, these same treatments suppress GnRH or LH secretion (reviewed in Reference 9). During fasting, another type of stressor, the increase of LH in response to NK3R agonist treatment is even greater in pubertal rats compared with ad libitum feeding (10). In contrast, responses to kisspeptin or dynorphin or agonists that bind to their cognate receptors, G protein-coupled receptor 54 and -opioid receptor (KOR), respectively, are more consistent across the reproductive cycle (8). In this issue, Grachev et al (11) report the results of a series of experiments that link the suppression of reproduction by immune/inflammatory stress with the NKB/ NK3R pathway in female rats. They first determined whether NK3R or KOR, the receptor for which dynorphin has the greatest affinity, were necessary for the LPS-induced suppression of LH pulses or increases in corticosterone (CORT) concentrations (11). Administration of the KOR antagonist nor-BNI did not reverse the effects of LPS on either LH pulsatility or CORT secretion, whereas the NK3R antagonist (SB222200) did reverse the suppression of LH pulsatility, although CORT remained elevated (11). This suggests that immune/inflammatory stress acts upstream of NKB to increase CORT secretion, and the effects of CORT on LH pulsatility are at least in part mediated through NK3R-containing neurons (11). Administration of LPS stimulates other factors involved in the immune/inflammatory response, including prostaglandins and ILs (12). In sheep, blocking prostaglandin synthesis reverses the LPS-induced suppression of LH in the luteal (6) but not the follicular phase (5). In women with polycystic ovary syndrome, a disorder including failure to ovulate and development of large cystic

Prenatal androgen excess enhances stimulation of the GNRH pulse in pubertal female rats.

In adolescent girls with polycystic ovary syndrome (PCOS), neuroendocrine derangements manifest after the onset of puberty, characterized by rapid LH pulse frequency. The early mechanism underlying the pubertal regulation of the GNRH/LH pulsatile release in adolescents with PCOS remains uncertain. To determine the effects of prenatal androgen exposure on the activation of GNRH neurons and generation of LH pulse at puberty, we administrated 5α-dihydrotestosterone to pregnant rats and observed serum LH levels and expression of hypothalamic genes in female offspring from postnatal 4 to 8 weeks. The 6-week-old prenatally androgenized (PNA) female rats exhibited an increase in LH pulse frequency. The hypothalamic expression of neurokinin B (Nkb (Tac2)) and Lepr mRNA levels in PNA rats increased remarkably before puberty and remained high during puberty, whereas elevated Kiss1 mRNA levels were detected only after the onset of puberty. Exogenous kisspeptin, NK3R agonist, and leptin triggered tonic stimulation of GNRH neurons and increased LH secretion in 6-week-old PNA rats. Leptin upregulated Kiss1 mRNA levels in the hypothalamus of pubertal PNA rats; however, pretreatment with a kisspeptin antagonist failed to suppress the elevated serum LH stimulated by leptin, indicating that the stimulatory effects of leptin may be conveyed indirectly to GNRH neurons via other neural components within the GNRH neuronal network, rather than through the kisspeptin-GPR54 pathway. These findings validate the hypotheses that NKB and leptin play an essential role in the activation of GNRH neurons and initiation of increased LH pulse frequency in PNA female rats at puberty and that kisspeptin may coordinate their stimulatory effects on LH release.

Blunted day-night changes in luteinizing hormone pulse frequency in girls with obesity: the potential role of hyperandrogenemia.

Puberty is marked by sleep-associated changes in LH pulse frequency and amplitude. Early pubertal girls with obesity exhibit blunted day-to-night changes in LH secretion; whether this occurs in late pubertal obese girls is unknown. The objective of the study was to test two hypotheses: 1) blunted day-to-night changes in LH secretion occur in both early and late pubertal obese girls, and 2) such alterations are specifically associated with hyperandrogenemia. This was a cross-sectional analysis. The study was conducted at a clinical research center. Twenty-seven early pubertal, premenarcheal girls (12 of whom were obese) and 63 late pubertal (postmenarcheal) girls (27 of whom were obese) participated in the study. Blood samples were taken every 10 minutes from 7:00 pm to 7:00 am. Change in LH pulse frequency [LH interpulse interval (IPI)] from daytime hours (7:00 pm-11:00 pm, while awake) to nighttime hours (11:00 pm to 7:00 am, while generally asleep). Both nonobese and obese postmenarcheal girls demonstrated significant day-to-night decreases in LH pulse frequency (IPI increases of 33% and 16%, respectively), but day-to-night changes were blunted in obese girls (P = .004, obese vs nonobese). Day-to-night LH pulse frequency decreased significantly in postmenarcheal obese subjects with normal T concentrations (26% IPI increase) but not in those with hyperandrogenemia. Similar differences were evident for LH pulse amplitude. Nonobese and obese early pubertal girls exhibited nonsignificant differences in day-night LH pulse frequency (day to night IPI increase of 26% vs decrease of 1%, respectively). Day-to-night changes in LH pulse secretion are blunted in postmenarcheal obese adolescent girls. This phenomenon may in part reflect hyperandrogenemia.

Neurokinin B Signaling in the Female Rat: a Novel Link Between Stress and Reproduction

Acute systemic stress disrupts reproductive function by inhibiting pulsatile gonadotropin secretion. The underlying mechanism involves stress-induced suppression of the GnRH pulse generator, the functional unit of which is considered to be the hypothalamic arcuate nucleus kisspeptin/neurokinin B/dynorphin A neurons. Agonists of the neurokinin B (NKB) receptor (NK3R) have been shown to suppress the GnRH pulse generator, in a dynorphin A (Dyn)-dependent fashion, under hypoestrogenic conditions, and Dyn has been well documented to mediate several stress-related central regulatory functions. We hypothesized that the NKB/Dyn signaling cascade is required for stress-induced suppression of the GnRH pulse generator. To investigate this ovariectomized rats, iv administered with Escherichia coli lipopolysaccharide (LPS) following intracerebroventricular pretreatment with NK3R or κ-opioid receptor (Dyn receptor) antagonists, were subjected to frequent blood sampling for hormone analysis. Antagonism of NK3R, but not κ-opioid receptor, blocked the suppressive effect of LPS challenge on LH pulse frequency. Neither antagonist affected LPS-induced corticosterone secretion. Hypothalamic arcuate nucleus NKB neurons project to the paraventricular nucleus, the major hypothalamic source of the stress-related neuropeptides CRH and arginine vasopressin (AVP), which have been implicated in the stress-induced suppression of the hypothalamic-pituitary-gonadal axis. A separate group of ovariectomized rats was, therefore, used to address the potential involvement of central CRH and/or AVP signaling in the suppression of LH pulsatility induced by intracerebroventricular administration of a selective NK3R agonist, senktide. Neither AVP nor CRH receptor antagonists affected the senktide-induced suppression of the LH pulse; however, antagonism of type 2 CRH receptors attenuated the accompanying elevation of corticosterone levels. These data indicate that the suppression of the GnRH pulse generator by acute systemic stress requires hypothalamic NKB/NK3R signaling and that any involvement of CRH therewith is functionally upstream of NKB.

The kisspeptin-GnRH pathway in human reproductive health and disease

BACKGROUNDThe discovery of kisspeptin as key central regulator of GnRH secretion has led to a new level of understanding of the neuroendocrine regulation of human reproduction. The related discovery of the kisspeptin-neurokinin B-dynorphin (KNDy) pathway in the last decade has further strengthened our understanding of the modulation of GnRH secretion by endocrine, metabolic and environmental inputs. In this review, we summarize current understanding of the physiological roles of these novel neuropeptides, and discuss the clinical relevance of these discoveries and their potential translational applications.METHODSA systematic literature search was performed using PUBMED for all English language articles up to January 2014. In addition, the reference lists of all relevant original research articles and reviews were examined. This review focuses mainly on published human studies but also draws on relevant animal data.RESULTSKisspeptin is a principal regulator of the secretion of gonadotrophins, and through this key role it is critical for the onset of puberty, the regulation of sex steroid-mediated feedback and the control of adult fertility. Although there is some sexual dimorphism, both neuroanatomically and functionally, these functions are apparent in both men and women. Kisspeptin acts upstream of GnRH and, following paracrine stimulatory and inhibitory inputs from neurokinin B and dynorphin (KNDy neuropeptides), signals directly to GnRH neurones to control pulsatile GnRH release. When administered to humans in different isoforms, routes and doses, kisspeptin robustly stimulates LH secretion and LH pulse frequency. Manipulation of the KNDy system is currently the focus of translational research with the possibility of future clinical application to regulate LH pulsatility, increasing gonadal sex steroid secretion in reproductive disorders characterized by decreased LH pulsatility, including hypothalamic amenorrhoea and hypogonadotropic hypogonadism. Conversely there may be scope to reduce the activity of the KNDy system to reduce LH secretion where hypersecretion of LH adds to the phenotype, such as in polycystic ovary syndrome.CONCLUSIONSKisspeptin is a recently discovered neuromodulator that controls GnRH secretion mediating endocrine and metabolic inputs to the regulation of human reproduction. Manipulation of kisspeptin signalling has the potential for novel therapies in patients with pathologically low or high LH pulsatility.

The Physiological Role of Arcuate Kisspeptin Neurons in the Control of Reproductive Function in Female Rats

Kisspeptin plays a pivotal role in pubertal onset and reproductive function. In rodents, kisspeptin perikarya are located in 2 major populations: the anteroventral periventricular nucleus and the hypothalamic arcuate nucleus (ARC). These nuclei are believed to play functionally distinct roles in the control of reproduction. The anteroventral periventricular nucleus population is thought to be critical in the generation of the LH surge. However, the physiological role played by the ARC kisspeptin neurons remains to be fully elucidated. We used bilateral stereotactic injection of recombinant adeno-associated virus encoding kisspeptin antisense into the ARC of adult female rats to investigate the physiological role of kisspeptin neurons in this nucleus. Female rats with kisspeptin knockdown in the ARC displayed a significantly reduced number of both regular and complete oestrous cycles and significantly longer cycles over the 100-day period of the study. Further, kisspeptin knockdown in the ARC resulted in a decrease in LH pulse frequency. These data suggest that maintenance of ARC-kisspeptin levels is essential for normal pulsatile LH release and oestrous cyclicity.

Increasing LH Pulsatility in Women With Hypothalamic Amenorrhoea Using Intravenous Infusion of Kisspeptin-54

Background:Hypothalamic amenorrhea (HA) is the one of the most common causes of period loss in women of reproductive age and is associated with deficient LH pulsatility. High-dose kisspeptin-54 acutely stimulates LH secretion in women with HA, but chronic administration causes desensitization. GnRH has paradoxical effects on reproductive activity; we therefore hypothesized that a dose-dependent therapeutic window exists within which kisspeptin treatment restores the GnRH/LH pulsatility in women with HA.Aim:The aim of the study was to determine whether constant iv infusion of kisspeptin-54 temporarily increases pulsatile LH secretion in women with HA.Methods:Five patients with HA each underwent six assessments of LH pulsatility. Single-blinded continuous iv infusion of vehicle or kisspeptin-54 (0.01, 0.03, 0.10, 0.30, or 1.00 nmol/kg/h) was administered. The LH pulses were detected using blinded deconvolution.Results:Kisspeptin increased LH pulsatility in all patients with HA, with peak responses observed at different doses in each patient. The mean peak number of pulses during infusion of kisspeptin-54 was 3-fold higher when compared with vehicle (number of LH pulses per 8 h: 1.6 ± 0.4, vehicle; 5.0 ± 0.5, kisspeptin-54, P < .01 vs vehicle). The mean peak LH pulse secretory mass during kisspeptin-54 was 6-fold higher when compared with vehicle (LH pulse secretory mass in international units per liter: 3.92 ± 2.31, vehicle; 23.44 ± 12.59, kisspeptin-54; P < .05 vs vehicle).Conclusions:Kisspeptin-54 infusion temporarily increases LH pulsatility in women with HA. Furthermore, we have determined the dose range within which kisspeptin-54 treatment increases basal and pulsatile LH secretion in women with HA. This work provides a basis for studying the potential of kisspeptin-based therapies to treat women with HA.

Parturition to resumption of ovarian cyclicity: comparative aspects of beef and dairy cows

There is a variable anoestrous period following parturition in the cow. Follicular growth generally resumes within 7 to 10 days in the majority of cows associated with a transient FSH rise that occurs within 3 to 5 days of parturition. Dairy cows that are not nutritionally stressed generally ovulate their first postpartum dominant follicle (~15 days), whereas beef suckler cows in good body condition normally have a mean of 3.2±0.2 dominant follicles (~30 days) to first ovulation; moreover, beef cows in poor body condition have a mean of 10.6±1.2 dominant follicles (~70 to 100 days) to first ovulation. The lack of ovulation of dominant follicles during the postpartum period is associated with infrequent LH pulses, with both maternal–offspring bonding and low body condition score (BCS) at calving being implicated as the predominant causes of delayed resumption of cyclicity in nursed beef cows. In dairy cows, the normal pattern of early resumption of ovulation may be delayed in high-yielding Holstein type cows generally owing to the effects of severe negative energy balance, dystocia, retained placental membranes and uterine infections. First ovulation, in both dairy and beef cows, is generally silent (i.e., no behavioural oestrus) and followed by a short inter-ovulatory interval (>70%). The key to optimizing the resumption of ovulation in both beef and dairy cows is appropriate pre-calving nutrition and management so that cows calve down in optimal body condition (BCS; 2.75 to 3.0) with postpartum body condition loss restricted to <0.5 BCS units.

Follicle size and reproductive hormone profiles during a post-weaning altrenogest treatment in primiparous sows

This study investigated the endocrine background of follicle size changes during post-weaning altrenogest treatment. altrenogest-treated sows received a 20-mg dosage daily at 8.00 a.m. from Day -1 to Day 14 after weaning. On Day -1, only 3/13 altrenogest-treated sows showed LH pulses compared with 8/8 control sows (P=0.001). On Day 0, control sows showed a typical high frequency-low amplitude LH pattern, indicative for recruitment of oestrogenic follicles. In altrenogest-treated animals on Day 0, half of the sows showed high frequency-high amplitude pulses from 4-5h after weaning. In altrenogest-treated sows, average follicle size increased from 3.1±0.5 mm on Day 0 to 4.4±0.6mm on Day 5, then decreased to 3.7±0.5 mm on Day 7 and stabilised thereafter. FSH and oestradiol (E2) concentrations showed a distinct diurnal pattern; high at 7.00 a.m. and low at 3.00 p.m. E2 concentrations (7.00 a.m.) showed a 2.5-fold increase from Day -1 to Day 2, and subsequently a 2-fold decline to reach a plateau at Day 8. FSH concentrations reached maximum levels by Day 5 and slowly declined afterwards. In conclusion, once-daily administration of altrenogest starting one day before weaning delays the weaning-induced increase in LH pulses. Although FSH and follicle size increase until Day 5 after weaning, follicle E2 production already decreased from Day 2 after weaning. Post-weaning altrenogest treatment thus results in a follicular wave of follicles that lose oestrogenic competence at Day 2 after weaning, presumably related to the changed LH dynamics during altrenogest treatment.

Exposure to the endocrine disruptor di(2-ethylhexyl)phthalate affects female reproductive features by altering pulsatile LH secretion

The patterns of growth of preovulatory follicles and corpora lutea, as well as plasma concentrations of estradiol, progesterone and LH were evaluated in sheep exposed to DEHP. There were not found effects on the preovulatory follicular dynamics nor on the ovulatory efficiency between DEHP-exposed and control sheep. However, plasma estradiol concentration was significantly higher in the ewes exposed to DEHP than in the control females (P<0.001). Afterwards, DEHP-exposed ewes had significantly higher plasma progesterone concentration from Day 2 of the luteal phase (P<0.05), although there were no differences between groups in the macro- and microscopic features of the corpora lutea. Analysis of mean basal concentrations of LH showed lower values in DEHP-exposed than in control sheep (0.3±01ng/mL vs. 0.7±0.1; P<0.05). Moreover, the frequency of LH pulses was 0.3pulses/4h, with amplitude of 0.6ng/mL, in the control group; on the other hand, the pulsatile secretion of LH in the DEHP-exposed group was undetectable during the period of sampling.

Kisspeptin, Neurokinin B, and Dynorphin Act in the Arcuate Nucleus to Control Activity of the GnRH Pulse Generator in Ewes

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists to GnRH receptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion. We thus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles for NKB and dynorphin within the KNDy neural network and raise the possibility that kisspeptin contributes to the control of GnRH pulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.

Evidence that Orphanin FQ Mediates Progesterone Negative Feedback in the Ewe

Orphanin FQ (OFQ), a member of the opioid family, is found in many areas of the hypothalamus and, when given centrally OFQ inhibits episodic LH secretion in rodents and sheep. Because GnRH neurons are devoid of the appropriate receptors to mediate steroid negative feedback directly, neurons that release OFQ may be involved. Using immunocytochemistry, we first determined that most OFQ neurons in the arcuate nucleus (ARC) and other hypothalamic regions of luteal phase ewes contained both estrogen receptor α and progesterone (P) receptor. Given a similar high degree of steroid receptor colocalization in other ARC subpopulations, we examined whether OFQ neurons of the ARC contained those other neuropeptides and neurotransmitters. OFQ did not colocalize with kisspeptin, tyrosine hydroxylase, or agouti-related peptide, but all ARC OFQ neurons coexpressed proopiomelanocortin. To test for a role for endogenous OFQ, we examined the effects of an OFQ receptor antagonist, [Nphe1,Arg14,Lys15]Nociceptin-NH2 (UFP-101) (30 nmol intracerebroventricular/h), on LH secretion in steroid-treated ewes in the breeding season and ovary-intact ewes in anestrus. Ovariectomized ewes with luteal phase concentrations of P and estradiol showed a significant increase in LH pulse frequency during infusion of UFP-101 (4.5 ± 0.5 pulses/6 h) compared with saline infusion (2.6 ± 0.4 pulses/6 h), whereas ewes implanted with only estradiol did not. Ovary-intact anestrous ewes displayed no significant differences in LH pulse amplitude or frequency during infusion of UFP-101. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of P on GnRH/LH pulse frequency in sheep.

Influence of ovarian manipulation on reproductive endocrinology in polycystic ovarian syndrome and regularly cycling women

Little is known about the function of the ovarian neuronal network in humans. In many species, copulation influences endocrinology through this network. As a first step, the possible influence of ovarian mechanical manipulation on pituitary and ovarian hormones was evaluated in polycystic ovarian syndrome (PCOS) and regularly cycling women. Prospective case-control study (2008-2010). Ten PCOS women (Rotterdam criteria) undergoing ovulation induction with recombinant-FSH and ten normal ovulatory controls were included in an academic fertility clinic. In the late follicular phase blood was drawn every 10 min for 6 h. After 3 h the ovaries were mechanically manipulated by moving a transvaginal ultrasound probe firmly over each ovary ten times. Main outcome measures were LH and FSH pulsatility and ovarian hormones before and after ovarian manipulation. All PCOS patients showed an LH decline after the ovarian manipulation (before 13.0 U/l and after 10.4 U/l, P<0.01), probably based on a combination of a longer LH pulse interval and smaller amplitude (P=0.07). The controls showed no LH change (before 9.6 U/l and after 9.3 U/l, P=0.67). None of the ovarian hormones (estradiol, progesterone, anti-Müllerian hormone, inhibin B, androstenedione and testosterone) changed in either group. Ovarian mechanical manipulation lowers LH secretion immediately and typically only in preovulatory PCOS patients. The immediate LH change after the ovarian manipulation without any accompanying ovarian hormonal changes point to nonhormonal communication from the ovaries to the pituitary. A neuronal pathway from the ovaries communicating to the hypothalamic-pituitary system is the most reasonable explanation.

Metabolic status, gonadotropin secretion, and ovarian function during acute nutrient restriction of beef heifers1,2

The effect of acute nutritional restriction on metabolic status, gonadotropin secretion, and ovarian function of heifers was determined in 2 experiments. In Exp. 1, 14-mo-old heifers were fed a diet supplying 1.2 × maintenance energy requirements (1.2M). After 10 d, heifers were fed 1.2M or were restricted to 0.4 × maintenance requirements (0.4M; d 0). Heifers received PGF2α (25 mg, intramuscularly) on d -10, 0, and 10 to synchronize ovulation. After 30 d, 1.2M and 0.4M heifers were realimented to 1.2 M for 100 d. Blood samples were collected every other day from d 0 to 14 then 3 times weekly thereafter. Heifers in Exp. 2 were managed as in Exp. 1 except that animals were fitted with an indwelling jugular catheter and blood samples were collected at 10-min intervals for 8 h on d 9, 10, and 11. Concentrations of progesterone in plasma were used to quantify ovarian luteal function. All 1.2M heifers ovulated, whereas only 30% of 0.4M heifers ovulated in Exp. 1. Concentrations of NEFA were greater and concentrations of thyroxine and IGF-I were less (P < 0.05) in plasma of 0.4M heifers compared with 1.2M heifers. The size of dominant follicles in Exp. 1 was reduced (P < 0.05) in 0.4M compared with 1.2M heifers. Concentrations of IGF-I were increased and anovulatory heifers resumed ovarian cycles an average of 35 d after realimentation. Concentrations of insulin were greater (P < 0.05) in plasma of 1.2M compared with 0.4M heifers in Exp. 2. The frequency of LH pulses was reduced (P < 0.05) in 0.4M heifers on d 9, and FSH in plasma on d 11 was not influenced by treatment. Reduced concentrations of IGF-I in plasma of nutrient-restricted heifers were associated with the reduced size of dominant follicles and indicated a local effect of growth factors on follicles. The decreased LH pulse frequency of 0.4M heifers before luteolysis indicates that restriction of nutrients decreased LH support of follicle growth. A preovulatory increase in estradiol in plasma and an ovulatory surge of LH were not detected in nutrient restricted heifers that did not ovulate. It is concluded that restricting beef heifers to 0.4 × maintenance energy requirements reduced the availability of metabolic fuel and decreased metabolic hormones, resulting in changes within the reproductive neuroendocrine-ovarian axis that compromised the ability of the dominant follicle to secrete sufficient concentrations of estrogen to stimulate an ovulatory surge of LH.

Intratesticular action of kisspeptin in rhesus monkey (Macaca mulatta)

Kisspeptin-Kiss1R signalling in mammals has been implicated as an integral part of the reproductive cascade. Kisspeptinergic neurons upstream of GnRH neurons are involved in the activation of the hypothalamic GnRH pulse generator during pubertal onset. Thus, the major research focus has been on the central effects of kisspeptin. The demonstration of the presence of KissR expression in human testes suggests additional unknown actions of kisspeptin-KISS1R signalling at the distal component of the male reproductive axis. Here we explored the impact of kisspeptin at the testis in the adult male rhesus monkey. We employed the clamped monkey model to assess the intratesticular actions of kisspeptin. Plasma testosterone and LH levels were monitored in four adult male monkeys. The peripheral administration of human kisspeptin-10 (50 μg, iv bolus) caused a single LH pulse, which was followed by a robust increase in plasma testosterone levels sustained for at least 180 min. This response was abolished when kisspeptin was administered to GnRH receptor antagonist (acyline) pre-treated animals. However, kisspeptin administration significantly (P < 0.005) elevated hCG-stimulated testosterone levels in acyline pre-treated monkeys when compared with saline+ hCG treatment. These results revealed a novel peripheral facet of kisspeptin signalling.

Estradiol Negative and Positive Feedback in a Prenatal Androgen-Induced Mouse Model of Polycystic Ovarian Syndrome

Gonadal steroid hormone feedback is impaired in polycystic ovarian syndrome (PCOS), a common endocrine disorder characterized by hyperandrogenism and an associated increase in LH pulse frequency. Using a prenatal androgen (PNA)-treated mouse model of PCOS, we aimed to investigate negative and positive feedback effects of estrogens on the hypothalamic-pituitary axis regulation of LH. PNA-treated mice exhibited severely disrupted estrous cycles, hyperandrogenism, significantly reduced fertility, and altered ovarian morphology. To assess the negative feedback effects of estrogens, LH was measured before and after ovariectomy and after estradiol (E2) administration. Compared with controls, PNA-treated mice exhibited a blunted postcastration rise in LH (P < .001) and an absence of LH suppression after E2 administration. To assess E2-positive feedback, control and PNA-treated GnRH-green fluorescent protein transgenic mice were subjected to a standard ovariectomy with E2-replacement regimen, and both plasma and perfusion-fixed brains were collected at the time of the expected GnRH/LH surge. Immunocytochemistry and confocal imaging of cFos and green fluorescent protein were used to assess GnRH neuron activation and spine density. In the surged group, both control and PNA-treated mice had significantly increased LH and cFos activation in GnRH neurons (P < .05) compared with nonsurged animals. Spine density was quantified in cFos-positive and -negative GnRH neurons to examine whether there was an increase in spine density in cFos-expressing GnRH neurons of surged mice as expected. A significant increase in spine density in cFos-expressing GnRH neurons was evident in control animals; however, no significant increase was observed in the PNA-treated mice because spine density was elevated across all GnRH neurons. These data support that PNA treatment results in a PCOS-like phenotype that includes impaired E2-negative feedback. Additionally, although E2-positive feedback capability is retained in PNA mice, elevated GnRH neuron spine density may reflect altered synaptic regulation.

Role of LH in the progesterone increase during the bromocriptine-induced prolactin decrease in heifers

The luteotrophic effect of bromocriptine in heifers was studied to determine if the reported posttreatment increase in progesterone (P4) just before or at the beginning of luteolysis was attributable to loss of a luteolytic effect of prolactin (PRL) or to the stimulation of LH, a known luteotropin. Four treatment groups (n = 7) were used: control (Ct), bromocriptine (Bc; 16 mg/heifer), acyline (Ac; 3 μg/kg), and bromocriptine and acyline combined (BcAc). Bromocriptine (inhibitor of PRL) and acyline (antagonist of GnRH and therefore blocker of LH) were given at Hour 0 on Day 16 postovulation, and blood samples were taken hourly at Hours 0 to 8. Concentration of P4 was greater (P < 0.05) in the Bc group than in the Ct group at each of Hours 1 to 8. Concentration of LH increased (P < 0.05) between Hours 0 to 2 in the Bc group but not in the other three groups. The peak of the first posttreatment LH pulse occurred earlier in the Bc group than in the Ct group. Average concentration of PRL was lower (P < 0.05) and number of PRL pulses was less (P < 0.05) in the Bc group than in the Ct group. Acyline inhibited LH in the Ac and BcAc groups as indicated by a decrease (P < 0.05) in concentration between Hours 0 and 2 and a decrease (P < 0.001) in number of pulses/heifer during the 8 h. A decrease in PRL but not an increase in P4 and LH occurred in the BcAc group. Results supported the hypothesis that the P4 increase associated with PRL suppression by bromocriptine treatment is attributable to an increase in LH.

Evidence That Dopamine Acts via Kisspeptin to Hold GnRH Pulse Frequency in Check in Anestrous Ewes

Recent work has implicated stimulatory kisspeptin neurons in the arcuate nucleus (ARC) as important for seasonal changes in reproductive function in sheep, but earlier studies support a role for inhibitory A15 dopaminergic (DA) neurons in the suppression of GnRH (and LH) pulse frequency in the nonbreeding (anestrous) season. Because A15 neurons project to the ARC, we performed three experiments to test the hypothesis that A15 neurons act via ARC kisspeptin neurons to inhibit LH in anestrus: 1) we used dual immunocytochemistry to determine whether these ARC neurons contain D2 dopamine receptor (D2-R), the receptor responsible for inhibition of LH in anestrus; 2) we tested the ability of local administration of sulpiride, a D2-R antagonist, into the ARC to increase LH secretion in anestrus; and 3) we determined whether an antagonist to the kisspeptin receptor could block the increase in LH secretion induced by sulpiride in anestrus. In experiment 1, 40% of this ARC neuronal subpopulation contained D2-R in breeding season ewes, but this increased to approximately 80% in anestrus. In experiment 2, local microinjection of the two highest doses (10 and 50 nmol) of sulpiride into the ARC significantly increased LH pulse frequency to levels 3 times that seen with vehicle injections. Finally, intracerebroventricular infusion of a kisspeptin receptor antagonist completely blocked the increase in LH pulse frequency induced by systemic administration of sulpiride to anestrous ewes. These results support the hypothesis that DA acts to inhibit GnRH (and LH) secretion in anestrus by suppressing the activity of ARC kisspeptin neurons.

GPR54-Dependent Stimulation of Luteinizing Hormone Secretion by Neurokinin B in Prepubertal Rats

Kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.