Attenuated LH Surge Research Articles

Effects of mastitis on ovarian function and fertility in dairy cows

Summary. Mastitis has a deleterious effect on reproductive responses and fertility of dairy cows, which depends on whether it occurs before or after artificial insemination (AI). Subclinical intramammary infection (IMI) before AI reduced steroid concentrations in the preovulatory follicle of approximately one third of lactating cows, and was associated with low expression of major steroidogenic genes. Consequently, IMI induced an attenuated LH surge and delayed ovulation in 30% of cows with subclinical IMI; the remaining 70% exhibited normal responses. The reason for the diversity in reproductive responses of individual cows to subclinical IMI is unclear. Mastitis induced by Gram-negative or Gram-positive bacteria disrupted the developmental competence of the pool of oocytes at the germinal vesicle stage, resulting in low blastocyst rates. The specific immune/inflammatory molecules involved in impairment of reproductive responses in subclinical mastitic cows are poorly documented. Exposure of small antral follicles to subclinical mastitis induced by Gram-positive bacterial toxins had a long-term effect by reducing estradiol concentrations of preovulatory follicles. Unlike chronic subclinical mastitis, the disruptive effect of short-term clinical IMI before AI is time-dependent and involves lowered conception when IMI occurs close to the time of insemination. The effect of clinical intramammary infection on corpus luteum function is equivocal. In a recent study, inter-estrus interval and progesterone concentration were unaltered in most (95%) E. coli-mastitic cows treated with anti-inflammatory drugs. Fertility studies showed that fertility of subclinical mastitic cows is improved by the Ovsynch program, probably because of synchronized timing of ovulation relative to AI in cows that could otherwise exhibit delayed ovulation.

The Excitatory Peptide Kisspeptin Restores the Luteinizing Hormone Surge and Modulates Amino Acid Neurotransmission in the Medial Preoptic Area of Middle-Aged Rats

Reproductive success depends on a robust and appropriately timed preovulatory LH surge. The LH surge, in turn, requires ovarian steroid modulation of GnRH neuron activation by the neuropeptide kisspeptin and glutamate and gamma-aminobutyric acid (GABA) neurotransmission in the medial preoptic area (mPOA). Middle-aged females exhibit reduced excitation of GnRH neurons and attenuated LH surges under estrogen-positive feedback conditions, in part, due to increased GABA and decreased glutamate neurotransmission in the mPOA. This study tested the hypothesis that altered kisspeptin regulation by ovarian steroids plays a role in age-related LH surge dysfunction. We demonstrate that middle-aged rats exhibiting delayed and attenuated LH surges have reduced levels of Kiss1 mRNA in the anterior hypothalamus under estrogen-positive feedback conditions. Kisspeptin application directly into the mPOA rescues total LH release and the LH surge amplitude in middle-aged rats and increases glutamate and decreases GABA release to levels seen in the mPOA of young females. Moreover, the N-methyl-D-aspartate receptor antagonist MK801 blocks kisspeptin reinstatement of the LH surge. These observations suggest that age-related LH surge dysfunction results, in part, from reduced kisspeptin drive under estrogen-positive feedback conditions and that kisspeptin regulates GnRH/LH release, in part, through modulation of mPOA glutamate and GABA release.

Restoration of the Luteinizing Hormone Surge in Middle-Aged Female Rats by Altering the Balance of GABA and Glutamate Transmission in the Medial Preoptic Area1

Hypothalamic glutamate and gamma-aminobutyric acid (GABA) neurotransmission are involved in the ovarian hormone-induced GnRH-LH surge in rodents. We previously reported that middle-aged rats have significantly less glutamate release in the medial preoptic area than young rats on the day of the LH surge. The present study tested the hypothesis that the delayed and attenuated LH surge in ovariohysterectomized middle-aged rats primed with ovarian steroids results from reduced hypothalamic glutamate and increased GABA(A) neurotransmission. Microdialysis results show that middle-aged rats with attenuated LH surges had reduced extracellular glutamate and increased extracellular GABA levels in the medial preoptic area compared with young rats. Blocking GABA(A) receptors with bicuculline or inhibiting synaptic glutamate reuptake with L-trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular Glu in the medial preoptic area and partially restored LH surge amplitude in middle-aged rats without altering LH surge onset. Complete recovery of LH surge amplitude was observed in middle-aged rats treated with the combination of bicuculline and L-trans-pyrrolidine-2,4-dicarboxylic acid. This treatment also restored the extracellular glutamate:GABA ratio in the medial preoptic area of middle-aged rats to the level of young rats. Immunoblot analysis revealed that estradiol and progesterone treatment reduced SLC32A1(formerly known as vesicular GABA transporter) levels and increased SLC17A6 (formerly known as vesicular glutamate transporter 2) levels in the anterior hypothalamus of ovariohysterectomized young but not middle-aged rats. These data suggest that both reduced availability of glutamate and increased activation of GABA(A) receptors under estrogen-positive feedback conditions contribute to the age-related delay in onset and attenuated amplitude of the LH surge.

Attenuation of Preoptic Area Glutamate Release Correlates with Reduced Luteinizing Hormone Secretion in Middle-Aged Female Rats

Glutamate (Glu) and its receptors are involved in the maturation and maintenance of the neural mechanisms governing the preovulatory LH surge of young, reproductive-aged rodents and nonhuman primates. Little is known about the role of Glu in the delayed onset and reduced peak amplitude of the LH surge that characterizes female rodents during early reproductive senescence. The present study tested the hypothesis that the delayed and attenuated LH surge observed in middle-aged female rats is associated with altered hypothalamic Glu release. We used intracerebral microdialysis in young (3-4 months) and middle-aged (9-11 months) female rats to monitor changes in medial preoptic area Glu release and jugular vein catheters to monitor changes in serum LH levels. All animals were ovariectomized and injected with estradiol and progesterone in doses sufficient to produce a robust LH surge in most (approximately 70%) young rats. In both young and middle-aged females that surged, extracellular Glu levels were higher than in those that did not surge. Among animals that surged, the onset of the LH surge was significantly delayed, and the amplitude of the surge was significantly reduced in middle-aged compared with young rats. Middle-aged females also had significantly reduced extracellular Glu levels throughout the day of the LH surge when compared with young females. These data strongly suggest that age-related hypothalamic dysfunction contributes to reproductive aging independent of gonadal failure. We propose that reduced medial preoptic area Glu transmission contributes to reproductive aging by attenuating excitatory input to GnRH neurons.

Regulatory Role of Excitatory Amino Acids in Reproduction

Glutamate, the major excitatory amino acid (EAA) transmitter in the central nervous system, has been implicated as a critical mediator in brain function. Glutamate and its receptors are found in all key hypothalamic areas critically involved in reproduction. Administration of glutamate and its agonists can bring about LH release in animals with a steroid background. Antagonists of the ionotropic glutamate receptors inhibited LH release and abolished the steroid-induced and the preovulatory LH surge. Both NMDA and non-NMDA receptor antagonists can also inhibit pulsatile LH release in castrated animals. The preoptic area has been implicated as a primary site of action of NMDA, while non-NMDA agonists have been suggested to act primarily at the arcuate/median eminence level. While EAAs may act directly on GnRH neurons to enhance GnRH release, the majority of evidence suggests that an indirect mechanism, involving EAA activation of nitric oxide and/or catecholamines, plays a major role in the GnRH-releasing effects of EAAs. Furthermore, there is also some evidence that the tonic inhibitory effect of opioids on GnRH may also involve, at least in part, a suppression of glutamate. Finally, EAA stimulation of GnRH/LH release is markedly attenuated in middle-aged rats, suggesting that a defect in glutamate neurotransmission may underlie the attenuated LH surge observed in aging.

The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus: relationship to the luteinizing hormone surge and effects of age.

The morphometry of astrocytes in the arcuate nucleus exhibits cyclic changes during the estrous cycle leading to dynamic changes in the communication between neurotransmitters and neuropeptides that regulate pituitary hormone secretion. Data suggest that remodeling of direct and/or indirect inputs into GnRH neurons may influence the timing and/or amplitude of the preovulatory LH surge in young rats. We have previously found that aging alters the timing and amplitude of the LH surge. Therefore, the purpose of this study was to focus on the rostral preoptic area where GnRH cell bodies reside. We assessed the possibility that the morphometry of astrocytes in the rostral preoptic area displays time-related and age-dependent changes on proestrus. Our results demonstrate that, in young rats, astrocyte cell surface area decreases between 0800 h and 1200 h, before the initiation of the LH surge. Changes in surface area over the cycle were specific to astrocytes in close apposition to GnRH neurons. In contrast, in middle-aged rats astrocyte surface area was significantly less than in young rats and did not change during the day. These findings suggest that a loss of astrocyte plasticity could lead to the delayed and attenuated LH surge that has been previously observed in middle-aged rats.

Effects of aging on luteinizing hormone secretion, ovulation, and ovarian tissue-type plasminogen activator expression.

This study examined the effects of aging on LH surge magnitude, ovulation, and ovarian expression of tissue-type plasminogen activator (tPA), a protease implicated in follicular rupture. While mean LH levels and ovulation rates were similar in middle-aged cyclic and young groups, there was a significant correlation between peak LH levels and ovulation rates in individual rats, such that females with lower LH surges ovulated fewer ova. In a separate experiment, proestrous LH levels were characterized in young and middle-aged rats, followed by in situ hybridization analysis of ovarian tPA mRNA. In young proestrous rats, tPA expression was observed in thecal-interstitial cells and oocytes, but not granulosa cells, prior to the LH surge. After the LH surge, there was a marked increase in tPA mRNA levels in granulosa cells of preovulatory, but not smaller follicles, peaking at 0200 hr estrus. By 0500 hr estrus, ovarian tPA expression declined, and ovulation had occurred. In contrast, LH-induced follicular tPA mRNA levels were dramatically lower in middle-aged rats with attenuated LH surges, and persisting preovulatory follicles were common in ovaries of these females on estrus morning. These findings suggest that age-related declines in ovulatory function result in part from altered induction of ovarian tPA expression, likely due to decreased proestrous LH secretion.

Decreased gonadotropin-releasing hormone neurosecretory response to glutamate agonists in middle-aged female rats on proestrus afternoon: a possible role in reproductive aging?

Before becoming acyclic, middle-aged rats display an attenuated LH surge and a decreased number of activated GnRH neurons. The present study examined whether the decreased activation of GnRH neurons in middle-aged rats could be due to defective glutamate neurosignaling in the hypothalamus. Arcuate nucleus/median eminence (ARC/ME) fragments were isolated from young (2-month-old) and middle-aged (9- to 11-month-old) rats at 1700 h on proestrus and incubated in vitro with or without the specific glutamate agonists, D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (1 mM), kainate (1 mM), and N-methyl-D-aspartate (NMDA; 50 mM). The results showed that basal GnRH release was similar in the two age groups. In contrast, stimulated GnRH release by D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate, and NMDA was significantly attenuated in middle-aged vs. young rats. KCl stimulation at the end of the experiments confirmed the viability of all ARC/ME fragments. Quantitative reverse transcriptase-PCR revealed that messenger RNA levels for the major NMDA receptor subunit (NMDAR1) were significantly lower in the preoptic area and ARC/ME of the middle-aged rat on proestrus afternoon. As a whole, these findings suggest that a defect in hypothalamic glutamate neurosignaling may be an important mechanism leading to age-related defects in LH secretion and acyclicity in female animals.

Alterations in plasma and ovarian immunoreactive inhibin during reproductive aging in the female rat

Previously, we showed that ovarian inhibin α- and β;(A)-subunit mRNAs are elevated in middle-aged and old persistent-estrous (PE) female rats. To determine whether higher inhibin subunit mRNA expressions result in increased circulating inhibins during reproductive aging, plasma immunoreactive inhibin α (ir-inh α) and gonadotropins were measured in young, middle-aged and PE rats. Plasma LH profiles were distinctly divergent in the middle-aged rats with some showing LH surges indistinguishable from young rats and others showing significantly attenuated LH surges. Plasma ir-inh α in middle-aged rats with LH surges were similar to those of young rats. However, animals with attenuated LH surges had higher peak ir-inh α levels than young and middle-aged animals with LH surges. Immunohistochemistry revealed increased levels of ovarian inhibin α staining in those animals with attenuated LH surges. Overall, the highest plasma and ovarian inhibin α were found in PE rats which lack LH surges. However, significant decreases of plasma and ovarian inhibin α were seen after reinstatement of estrous cyclicity with progesterone implant treatment. Thus, increases in both plasma and ovarian inhibin α appear to be closely associated with attenuation or loss of the preovulatory gonadotropin surge that occurs during aging.

Luteinizing hormone secretion by dispersed anterior pituitary gland cells from middle-aged rats.

Several studies have shown that a certain subpopulation of middle-aged rats have an attenuated preovulatory LH surge, and that this is an early indicator of a general decline in reproductive cyclicity. The pituitary glands of middle-aged animals with attenuated LH surges have been shown to be significantly less responsive to GnRH than those from young (Y) animals. The present study was designed to investigate the relationship between the in vivo LH surge and the in vitro secretory capacity of dispersed anterior pituitary gland cells from the same animals. Individual LH-secreting cells were also studied with respect to their LH secretory capacity and proportionate numbers in the pituitary gland using reverse hemolytic plaque assay (RHPA). Two groups of animals, young (4-5 months old) and middle-aged (10-12 months old), were sampled on the afternoon of proestrus to determine the amplitudes of their preovulatory LH surges. The middle-aged animals were then classified as either normal (MAN) or attenuated (MAA) with regard to the peak levels of the surges. On the next proestrus, dispersed anterior pituitary gland cells were prepared for either perifusion or RHPA. Perifused anterior pituitary gland cells were exposed to three 2.5-min pulses of GnRH(30 nM) at 60-min intervals. In the RHPA, red blood cells were treated with protein-A to allow coating with rabbit antiovine LH-beta, and then co-incubated with anterior pituitary gland cells. The cells were then treated with one of several concentrations of GnRH (0.01, 1.0, 10, or 100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

A relative depletion of luteinizing hormone-releasing hormone was observed in the median eminence of young but not middle-aged rats on the evening of proestrus.

Computer-assisted analysis was used to examine LHRH reaction product in the median eminence of young and middle-aged rats prior to and after the expected peak of the LH surge on proestrus. The area of LHRH reaction product was analyzed in 5 rostral-caudal levels (A-E) of the median eminence. The relative depletion of LHRH in the median eminence of young females on the evening compared to the afternoon of proestrus suggested LHRH neurosecretion in conjunction with the preovulatory LH surge. The pattern of depletion observed further suggested that LHRH release may occur preferentially from restricted regions of the median eminence or in a coordinated wave-like pattern. Four of the five levels of the median eminence exhibited a relative decrease in LHRH on the evening of proestrus in young females, and this time-related difference in LHRH reaction product was statistically significant in median eminence levels B and C. In contrast, little evidence of a relative depletion in LHRH reaction product from early to late proestrus was observed in the median eminence of aging animals. Moreover, the concentration of the densest LHRH reaction product appeared diminished in the median eminence of middle-aged compared to young females at the time points examined in the present study. The age-related differences observed in LHRH reaction product in the median eminence may contribute to the attenuated LH surge documented in middle-aged female rats. Whether these changes in LHRH immunoreactivity can be attributed to age-related alterations in afferents received by LHRH neuronal cell bodies or terminals or to intrinsic deficits in signalling mechanisms within LHRH neurons remains to be determined. Computer-assisted analysis of the immunocytochemical data enabled the assessment of relative changes in reaction product within specific elements of LHRH neurons in precise regions of the median eminence.

Pro-Gonadotropin-Releasing Hormone (ProGnRH) and GnRH Content in the Preoptic Area and the Basal Hypothalamus of Anterior Medial Preoptic Nucleus/Suprachiasmatic Nucleus-Lesioned Persistent Estrous Rats*

The content of GnRH and its precursor peptide were quantified in female rats bearing lesions in the anterior medial preoptic nucleus (AMPO) and the suprachiasmatic nucleus (SCN), and the effects of the lesions on the synthetic activity of the GnRH neurons were evaluated. Electrolytic lesions which induced persistent estrous (PE), or irregular estrous cycles, were produced by passing 5-10 microA of direct current into the AMPO or the SCN of female rats which exhibited regular 4 days estrous cycles before the lesions. Approximately 5 weeks after lesion placement, blood samples were withdrawn from catheterized, freely moving animals and plasma LH, PRL, estrogen, and progesterone were determined by RIA. The preovulatory surges of LH and PRL were eliminated in AMPO- or SCN-lesioned PE rats. Moreover, the LH surge was eliminated and the PRL surge significantly attenuated after estrogen and progesterone treatment of rats bearing complete lesions, irrespective of the presence of ovaries. Irregular cycling animals with incomplete AMPO or SCN lesions, exhibited attenuated LH surge and PRL surge similar to proestrous controls. In one incidence this occurred spontaneously, and could also be induced by sequential estrogen and progesterone injections. After ovariectomy, plasma LH levels were significantly lower in the lesioned animals as compared to sham operated rats (P less than 0.05). Similar secretory patterns of LH and PRL were obtained from a second series of sham-operated rats during the different stages of the estrous cycle or from AMPO- or SCN-lesioned rats during persistent estrus. After 2 months the animals were killed between 0830 and 0930 h, and the preoptic area and the basal hypothalamus were microdissected from the brain sections. After extraction and purification, proGnRH and GnRH levels were measured by RIA. ProGnRH levels in the preoptic area were significantly reduced in AMPO- or in SCN-lesioned rats, compared to proestrous controls (P less than 0.01). In contrast, GnRH levels in either area did not differ in AMPO- or in SCN-lesioned animals compared to sham-operated, proestrous controls. Therefore, lesions of the AMPO or the SCN produce PE and reduce proGnRH without reducing GnRH levels. These data would suggest that the AMPO and the SCN participate in the control of the estrous cycle and are necessary for preovulatory surges of PRL and LH to occur and that the AMPO and the SCN form part of the neural circuit that regulates GnRH synthesis and/or release.

Endogenous luteinizing hormone surge during superovulation induction with sequential use of clomiphene citrate and pulsatile human menopausal gonadotropin.

There is dispute as to whether an endogenous LH surge occurs in women during follicle stimulation with human menopausal gonadotropin (hMG) for in vitro fertilization (IVF). In this study, 12 consecutive normally cycling women with tubal infertility were treated with clomiphene citrate and hMG for ovulation induction and subsequent IVF. Beginning on the fifth day of clomiphene administration (150 mg daily for 5 days), 225 IU hMG were given daily sc in a pulsatile manner (28 IU/3 h). All women had an endogenous LH surge which started on the seventh to ninth day of hMG administration. The mean duration [25.5 +/- 1.0 (+/- sem) h] and the mean peak value (38.9 +/- 4.7 U/liter) of the LH surge were less than those in spontaneous cycles. However, luteinization of at least some of the follicles occurred, based on findings of supraphysiological levels of serum progesterone during the surge and subsequent increase in urinary pregnanediol levels (luteal phase). Preovulatory oocytes were recovered via laparoscopy 34-35 h after the onset of the surge, and these oocytes cleaved after IVF. One pregnancy was achieved after embryo transfer, which resulted in abortion. These results represent the first demonstration that an attenuated LH surge is obtained in normally cycling women during superovulation induction with sequential clomiphene/pulsatile hMG treatment.

Pituitary and gonadal response to exogenous LH-releasing hormone in the male domestic cat.

To determine the influence of exogenous LH-releasing hormone (LHRH) on serum LH and testosterone, ten adult male domestic cats received three treatments on a rotating schedule at 10-day intervals as follows: (I) 0.1 ml saline i.m. (control); (II) 10 micrograms LHRH i.m., single injection; (III) 10 micrograms LHRH i.m., two injections given at a 2-h interval. Serial blood samples collected over a 360-min interval were analysed by radio-immunoassay for LH and testosterone. Although baseline serum LH values in saline-treated animals (treatment I) varied markedly among individual cats (2.2-29.2 micrograms/l), there was no evidence of pulsatile LH release or alterations in testosterone over time within individual males. In treatment II, the single injection of LHRH induced a rapid rise in mean serum LH within 30 min in all cats (mean peak, 88.2 +/- 9.8 micrograms/l), which returned to baseline by 120 min after LHRH. Mean testosterone increased within 30 min in this group (from 6.03 +/- 2.18 to 18.55 +/- 3.36 nmol/l), peaked at the 60-min collection (19.76 +/- 2.77 nmol/l) and returned to baseline by the 150-min sample. After treatment III, serum LH peaked at 131.6 +/- 13.6 micrograms/l within 30 min of the initial LHRH injection. A second injection of LHRH produced another LH surge within 30 min, but in all cats this second response was of a lower magnitude (mean peak, 69.0 +/- 14.5 micrograms/l) and shorter duration (P less than 0.05). The second LHRH injection sustained peripheral testosterone levels for approximately 1 additional h.(ABSTRACT TRUNCATED AT 250 WORDS)