Anti-Müllerian hormone-mediated preantral follicle atresia is a key determinant of antral follicle count in mice.

STUDY QUESTION: Does anti-Müllerian hormone (AMH) induce preantral follicle atresia in mice? SUMMARY ANSWER: The present findings suggest that AMH-mediated follicle atresia only occurs in early follicles before they become sensitive to FSH. WHAT IS KNOWN ALREADY: Most prior studies have investigated the ability of AMH to inhibit primordial follicle activation. Our previous study showed that AMH-overexpressing mice had fewer preantral follicles than expected after accounting for primordial follicle inhibition but the reason for this was not determined. STUDY DESIGN, SIZE, DURATION: Cross-sectional-control versus transgenic/knockout mouse studies were carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies were conducted on female wild-type (Amh+/+), AMH-knockout (Amh-/-) and AMH overexpressing (Thy1.2-AMHTg/0) mice on a C57Bl/6J background (age: 42-120 days). The follicle counts were conducted for primordial, transitioning, primary, secondary and antral follicles in Amh-/- and Amh+/+ mice. After confirming that follicle development speeds were identical (proliferating cell nuclear antigen immunohistochemistry), the ratio of follicles surviving beyond each stage of folliculogenesis was determined in both genotypes. Evidence for increased rates of preantral follicle atresia was assessed by active caspase-3 immunohistochemistry in wild-type and Thy1.2-AMHTg/0 mice. MAIN RESULTS AND THE ROLE OF CHANCE: Amh -/- mice at 100-120 days of age had lower primordial follicle counts but higher primordial follicle activation rates compared to Amh+/+ mice. These counteracting effects led to equivalent numbers of primordial follicles transitioning to the primary stage in Amh+/+ and Amh-/- mice. Despite this, Amh+/+ mice had fewer primary, secondary, small antral and medium antral follicles than Amh-/- mice indicating differing rates of developing follicle atresia between genotypes. Cleaved caspase-3 immunohistochemistry in Thy1.2-AMHTg/0 ovaries revealed high rates of granulosa cell and oocyte apoptosis in late primary/early secondary follicles of Thy1.2-AMHTg/0 mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The findings were shown only in one species and additional research will be required to determine generalizability to other species. WIDER IMPLICATIONS OF THE FINDINGS: This study is consistent with prior studies showing that Amh-/- mice have increased primordial follicle activation but these new findings demonstrate that AMH-mediated preantral follicle atresia is a predominant cause of the increased small antral follicle counts in Amh-/- mice. This suggests that the role of AMH is not to conserve the ovarian reserve to prolong fertility, but instead to prevent the antral follicle pool from becoming too large. While this study may demonstrate a new function for AMH, the biological purpose of this function requires further investigation, particularly in mono-ovulatory species. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Health Research Council of New Zealand and the University of Otago. No competing interests to declare.

Continuous low-dose treatment with brain-derived neurotrophic factor or neurotrophin-3 protects striatal medium spiny neurons from mild neonatal hypoxia/ischemia: a stereological study.

This study aimed to investigate whether continuous, low-dose, intracerebral infusion of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) could protect against striatal neuronal loss in mild neonatal hypoxic/ischaemic brain injury. Continuous, low-dose, intracerebral treatment is likely to minimise unwanted side effects of a single high dose and lengthen the time window for neuroprotection. A milder, albeit brain damage-inducing, hypoxic/ischaemic injury paradigm was used since this situation is likely to produce the highest survival rates and thus the greatest prevalence. Anaesthetised postnatal day 7 rats were each stereotaxically implanted with a brain infusion kit connected to a micro-osmotic pump. The pump continuously infused either BDNF (4.5 microg/day), NT-3 (12 microg/day), or vehicle solution into the right striatum for 3 days from postnatal day 7. The intrastriatal presence of BDNF or NT-3 was verified immunohistochemically. On postnatal day 8, the rats underwent right common carotid artery ligation followed by hypoxic exposure for 1.5 h. Animals were weighed daily thereafter and killed 1 week later on postnatal day 14. The total number of medium spiny neurons within the right striatum was stereologically determined using an optical disector/Cavalieri combination. Other measures of neuroprotection such as brain weight and striatal infarct volume were also undertaken. BDNF or NT-3 significantly increased the total number of surviving medium spiny neurons by 43% and 33% respectively. This significant neuroprotection was not evident when brain weight, striatal volume, striatal infarct volume, and neuronal density measures for NT-3, were compared. These measures therefore missed the protective effect demonstrated by the total neuronal count. This suggests that stereological measurement of total neuronal number is needed to detect neuroprotection at 1 week after low-dose, continuously infused, neurotrophin treatment and mild hypoxic/ischaemic injury. The results also suggest that lower treatment doses may be more useful than previously thought. BDNF may be particularly useful since it fostered both neuroprotection and normal weight gain. The ability to rescue striatal neurons from death may contribute toward a potential short-term, low-dose neurotrophin treatment for mild perinatal hypoxic/ischaemic brain injury in humans.

Is Fos protein expressed by dying striatal neurons after immature hypoxic-ischemic brain injury?

The transient induction of mRNA for the immediate-early gene c-fos has been reported following hypoxic-ischemic brain injury in the immature brain. However, no studies have examined the temporal expression of Fos protein, which is the functionally relevant product of c-fos gene expression. Increased expression of Fos protein has been linked to cell death. We therefore examined whether Fos protein is expressed by dying neurons after immature hypoxic-ischemic brain injury. A well characterized immature rat model of hypoxic-ischemic injury at postnatal day (PN) 7 was used. Three hypoxic-ischemic and three normoxic control pups were studied per time point (i.e., 0, 2, 12, 24, 48, and 72 h posttreatment). Expression of Fos within striatal and other neurons was detected immunocytochemically. Fos protein was expressed within dying striatal neurons at 0-12 h after hypoxia-ischemia. However, detection was only seen in 2 of 17 hypoxic-ischemic pups. These 2 pups had >/=80% of their striatal neurons dying within their right, hypoxic-ischemic-exposed hemisphere. Fos protein expression after severe injury may, therefore, be a response to extraordinary or extreme stress. The absence of Fos protein expression in the majority of hypoxic-ischemic pups, which all exhibited striatal neuronal death, suggests that Fos expression is not necessary for cell death to occur. Therapies directed against Fos protein expression may therefore have limited usefulness in immature hypoxic-ischemic brain injury.

Cerebral palsy and experimental hypoxia-induced perinatal brain injury: is magnesium protective?

Recent retrospective, observational studies describe an association between maternal administration of magnesium and a reduction in cerebral palsy in low birthweight infants. Cerebral palsy is thought to result mainly from hypoxia-induced brain injury, and its incidence is increasing due to recent advances in obstetric and newborn intensive care. The findings of the observational studies raise the promising possibility that magnesium treatment may protect the immature brain from hypoxia-induced injury and thus decrease the incidence of cerebral palsy. Despite this promise, a critique of clinical studies indicates that a definitive answer is dependent on the outcome of prospective clinical trials. A critique of experimental animal studies indicates that a neuroprotective role for magnesium prior to, or after, immature hypoxic-ischemic brain injury is currently not supported by the available evidence. Most clinical and animal studies in this field have used magnesium sulphate as a possible therapeutic agent. Based on evidence that magnesium chloride is a preferable salt to use, the inclusion of magnesium chloride in future clinical and experimental studies is worthy of consideration.

Postinjury magnesium sulfate treatment is not markedly neuroprotective for striatal medium spiny neurons after perinatal hypoxia/ischemia in the rat.

Hypoxic/ischemic (H/I) brain injury is thought to be mediated via the N-methyl-D-aspartate receptor complex, which can be blocked by the magnesium ion. Striatal medium spiny neurons abundantly express N-methyl-D-aspartate receptors and are known to be injured after H/I. Thus, the aim of this study was to investigate the effect of postinjury magnesium treatment on the total number of medium spiny neurons in the striatum after perinatal H/I injury in the rat. Anesthetized postnatal day (PN) 7 rats underwent common carotid artery ligation followed 2 h later by exposure to hypoxia for 1.5 h. Contralateral hemispheres served as controls as did animals exposed to normoxia. Immediately after hypoxia or normoxia, the magnesium groups received s.c. injections of 300 mg/kg MgSO4. Control, hypoxic or normoxic animals received NaCl injections. This continued daily until PN13. Eleven matched-for-weight H/I pups were injected in total. A power calculation showed that 11 pups per treatment group would permit detection of a treatment difference of 32% or more. Animals were killed on PN18, and 40-micron serial sections were cut through each entire striatum. The total number of the predominant medium spiny neurons within each striatum was stereologically determined via the use of an unbiased optical dissector/Cavalieri combination. It was found that postinjury magnesium treatment did not improve neuronal survival by 32% or more in the striatum. The results suggest that magnesium treatment after perinatal H/I damage in the rat is not markedly neuroprotective for striatal medium spiny neurons.

The trophic requirements of mature motoneurons.

Mature motoneurons interact with many cell types, including skeletal muscle fibres, Schwann cells, glia and various neurons. Each of these cell types is thought to provide trophic support to motoneurons, but it is not known whether the support provided by one cell type can fully substitute for the absence of a signal from another cell type. The ability of various growth factors to support motoneurons in the absence of muscle fibres, Schwann cells or long-axon synaptic input has been studied using in vivo models. However, these studies do not define the total needs of motoneurons, as local spinal influences have not been removed. In this paper, the total trophic requirement of mature motoneurons was assessed by culturing them at a low cell density, in the absence of all other cell types. Under these conditions, mature motoneurons die by apoptosis within 24 h, which is equivalent to the rate at which immature motoneurons die in vitro. This is consistent with the emerging view that mature cells are primed for apoptosis. Nine putative trophic factors (BDNF, CNTF, FGF2, GDNF, IGF I, IGF II, NT3, NT4, TGF-beta2), either alone or in combination, were unable to prevent the rapid death of the cultured motoneurons, even though some of these factors are able to attenuate the affects of less severe injuries such as axotomy or avulsion. The survival of mature motoneurons may therefore be dependent on a combination of growth factors, with at least one of the factors being distinct from the above mentioned factors.

Do aluminium and/or glutamate induce Alz-50 reactivity? A light microscopic immunohistochemical study.

Alzheimer's disease is thought to be characterized by conformational and phosphorylation changes in tau protein, leading to the formation of aggregations of paired helical filaments within neurons. Potential agents for inducing conformational changes in tau, namely aluminium and glutamate, were investigated in this study. Explant cultures of cortical neurons were established from embryonic day 17 rat fetuses. Cultures were exposed to aluminium, glutamate, aluminium/glutamate, aluminium/citric acid and citric acid (since aluminium is thought to enter cells via the transferrin receptor by complexing with citric acid) from 7-12 days in vitro. Control explants were exposed to basal medium only. On day 12, explants were paraffin-embedded. Four-six explants were serially sectioned per condition. For each explant, every 10th and adjacent 4 microm section were randomly selected and processed, with controls, for: (1) alcoholic morin histochemistry (to detect intracellular aluminium), (2) Perls' iron histochemistry (to control for the morin stain which also detects iron), (3) neurofilament immunohistochemistry (to estimate total neuronal number per explant) and (4) Alz-50 immunohistochemistry (to detect possible conformational changes in tau). The absolute number of stained/immunoreactive neurons was determined per explant. The percentage incidence was then determined per explant and averaged per condition. Explants in the aluminium conditions contained significant increases in the incidence of morin-positive aluminium containing neurons. There was also a significant increase in the incidence of Alz-50 positive neurons for the aluminium compared with control explants. These results suggest: (1) aluminium enters neurons and (2) aluminium alone induces possible conformational changes in tau as detected by the Alz-50 antibody, while aluminium combined with glutamate, or glutamate alone, do not.

Do aluminium and/or glutamate induce Alzheimer PHF-like formation? An electron microscopic study.

Paired helical filaments (PHFs) constitute the majority of filaments in neurofibrillary tangles (NFTs), an Alzheimer's disease (AD) characteristic. PHFs consists of two filaments helically twisted around one another in a regular pattern. The effects of possible PHF-inducing candidates, namely aluminium and glutamate, were observed at the ultrastructural level in this investigation. Rat cerebral explants were exposed to aluminium, citric acid and glutamate singly or combined from 7-12 days in vitro (DIV), while control explants remained in basal medium. On 12 DIV, explants were processed for EM. Three-four EM explants were serially sectioned per condition. Ten 60 nm sections from five systematically sampled areas per explant were collected. One section was randomly chosen per sampled area and all neurons within it observed at 81,200x to record the presence of accumulations of curved filaments (CFs), straight filaments (SFs) or PHFs. Using stereological methods, absolute numbers and the percentage incidence of CFs and SFs were calculated. A significant increase in the frequency of neurons containing CF aggregations in aluminium explants compared to glutamate explants was found. There were no significant differences between conditions for neurons containing SF accumulations. Possible PHFs were observed in one aluminium/glutamate-treated explant. These results suggest that aluminium alone can cause significant formation of accumulations of C- or S-shaped CFs, some of which are double-stranded and twisted around one another regularly. However, structures that were possibly PHF-like were only observed in one aluminium-treated explant, thus making it premature to draw an association between aluminium and the induction of AD-like pathology.

Total number of neurons in the neostriatal, pallidal, subthalamic, and substantia nigral nuclei of the rat basal ganglia: a stereological study using the cavalieri and optical disector methods.

The total number of neurons within six subdivisions of the rat basal ganglia was estimated using unbiased stereological counting methods and systematic random sampling techniques. Six young adult rats were perfuse-fixed, their right cerebral hemispheres were embedded in glycolmethacrylate, and a complete set of serial 40-mu m sections was cut through each hemisphere. After a random start, a systematic subset (e.g., every tenth) of these sections was used to estimate the total volume of each subdivision using Cavalieri's method. The same set of sampled sections was used to estimate the number of neurons in a known subvolume (i.e., the Nv) by the optical disector method. The product of the total volume and the Nv by these methods yields an unbiased estimate of the total number of neurons. It was found that the right basal ganglia consisted, on average, of 2.79 million neostriatal or caudate-putamen neurons (with a coefficient of variation of 0.07), 46,000 external globus pallidus neurons (0.11), 3,200 entopeduncular/internal globus pallidus neurons (0.10), 13,600 subthalamic neurons (0.10), 7,200 substantial nigra pars compacta neurons (0.15), and 26,300 substantia nigra pars reticulata neurons (0.07).

Embedding, sectioning, immunocytochemical and stereological methods that optimise research on the lesioned adult rat spinal cord.

Numerous methods have been developed to embed, section and immunocytochemically label nervous tissue and the method chosen depends upon numerous factors. However, many of these methods have technical drawbacks that make them difficult to use in studies using injured/lesioned tissue. We present here, methods for embedding, sectioning and immunocytochemically labelling lesioned adult spinal cord tissue at the light microscope level. We have developed a novel, gelatine-embedding technique for vibratome sectioning which overcomes many of the difficulties encountered with lesioned tissue. Individualised immunocytochemical protocols have also been developed for the antibodies GFAP (to label astrocytes), MBP (to label myelin) and CNP-ase (to label oligodendrocytes). Sequential pre-treatment with proteinase-K, methanol and sodium borohydride achieved optimal GFAP localisation. MBP and CNP-ase were optimally localised after sequential pre-treatment with proteinase-K (at different concentrations) and sodium borohydride. Methanol pre-treatment resulted in a loss of immunoreactivity for these latter two antibodies. Each protocol achieved full immunocytochemical penetration throughout 40 microns vibratome sections. These techniques enable the new unbiased stereological tools (that is, the Cavalieri and optical disector principles) to be utilised.

Age-related increase in the total number of corticotropin-releasing hormone neurons in the human paraventricular nucleus in controls and Alzheimer's disease: comparison of the disector with an unfolding method.

It has been hypothesized that the corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) become hyperactive with age, and even more so in Alzheimer's disease. This hyperactivity could be due to an increased production of CRH per neuron, or an increased number of PVN neurons producing CRH, or both. As a first step in elucidating which of these biological mechanisms might be operative, we have estimated the absolute number of CRH immunoreactive neurons in the PVN of 10 human control subjects between 36 and 91 years of age and 10 Alzheimer patients between 40 and 97 years of age. CRH neurons were immunocytochemically detected in 6 microns paraffin sections with the aid of a highly specific monoclonal antibody to CRH. The antibody signal was amplified by the biotin-streptavidin and alkaline phosphatase methods. The absolute number of CRH neurons in the PVN was obtained by multiplying the number of CRH neurons in a unit volume (NV) by the total volume of the PVN. Two different methods were used to estimate the NV: an unfolding method and a disector method (about three times more time-consuming). Compared to the disector, the unfolding method consistently yielded a lower cell number for all patients by 38% (+/- 2.8%; mean +/- SEM). However, both methods yielded an increase in the absolute number of CRH neurons in control and Alzheimer patients with age. No statistically significant difference in the absolute number of CRH neurons was found between control and Alzheimer patients with both methods. The age-dependent increase in the absolute number of CRH neurons within the PVN of both control and Alzheimer patients is interpreted as a sign of activation of the CRH neurons with age.

Neuronal survival and neurite growth in cultured cerebral explants: assessment of the effect of cytosine arabinoside using improved stereology.

Using improved stereological procedures, based on measurements of the total reference volume, the effect of cytosine arabinoside (AraC) on neurons and non-neuronal cells in rat cerebral explants has been reinvestigated. These analyses revealed that, in the AraC-treated explants, neurons support a significantly increased volume of neurites, while the volume of non-neuronal cells in the outgrowth zone is significantly decreased. The total number of neurons is similar in the control and treated explants. Since AraC primarily affects dividing cells, these data suggest that the increased volume of neurites per neuron may be mediated through a curtailment of non-neuronal cell division in the AraC-treated explants. This volumetric approach represents a major improvement on current morphometric approaches for quantifying cultured explants of nervous tissue, and provides important evidence that the previously postulated effects of AraC are real ones.

Effect of cytosine arabinoside on rat cerebral explants: fibroblasts and reactive microglial cells as the primary cellular barrier to neurite growth.

Rat cerebral explants were cultured in the presence or absence of the mitotic inhibitor cytosine arabinoside (AraC) to investigate whether reports of increased neurite outgrowth may be primarily related to a decreased incidence of fibroblastic-reactive microglial cells. Treated explants were exposed to AraC from 7 to 10 days in vitro (DIV) and processed for transmission electron microscopy at 18 DIV. Morphometric analysis of the outgrowth zone revealed a statistically significant decrease in the incidence of fibroblastic-reactive microglial cells (from 12 to 0%), and a significant increase in the incidence of protoplasmic astrocytic-epithelial cells (from 82 to 96%), for AraC-treated explants compared to controls. In contrast, the incidence of fibrous astrocytes was similar to that of control explants (4 and 6%, respectively). Thus, it appears that AraC may primarily enhance neurite growth by curtailing the proliferation of fibroblastic-reactive microglial cells. These results suggest that fibroblastic-reactive microglial cells, rather than fibrous astrocytes, may constitute the primary cellular barrier to neurite growth.

Effect of fluorodeoxyuridine on neurons and non-neuronal cells in cerebral explants.

Fetal rat brain neurons are reported to show enhanced neurite development when treated with the mitotic inhibitors fluorodeoxyuridine (FUdR) or cytosine arabinoside (AraC). For AraC-treated rat cerebral explants, increased neurite growth occurs along with a change in the composition of the non-neuronal cell population and diminished non-neuronal cell proliferation. FUdR-treated rat cerebral explants were therefore cultured in an attempt to determine whether FUdR encourages neurite outgrowth by changing the composition and number of non-neuronal cells. Quantitative morphological analyses revealed a significant decrease in the incidence of non-neuronal cells, and an increase in neurite outgrowth for the FUdR-treated explants. These explants also exhibited an increased proportion of protoplasmic astrocytic-epithelial cells and a decreased proportion of fibroblastic-reactive microglial cells. Thus, FUdR may encourage neurite outgrowth through a curtailment of non-neuronal cell proliferation (primarily by fibroblastic-reactive microglial cells) and through the creation of a non-neuronal cell environment consisting almost entirely of protoplasmic astrocytes.

Effect of cytosine arabinoside on the composition of the nonneuronal cell population in cerebral explants.

Rat cortical explants were cultured in the presence or absence of the mitotic inhibitor cytosine arabinoside to determine whether or not it affects the composition of the nonneuronal cell population within the outgrowth zone. Ultrastructural morphometric analysis of the incidence of fibroblasts, fibrous astrocytes, and protoplasmic astrocytes-epithelial cells at 18 days in vitro, revealed statistically significant decreases in the incidence of fibroblasts and fibrous astrocytes in the explants treated with the inhibitor compared with control explants. Coupled with earlier findings of enhanced neurite outgrowth and decreased nonneuronal cell proliferation that follows such treatment, it appears that cytosine arabinoside may potentiate neurite outgrowth by altering the composition, as well as the number, of nonneuronal cells in the outgrowth zone. These data indicate that fibroblasts and fibrous astrocytes may limit the regenerative response of severed axons in the mammalian central nervous system.

Non-neuronal cell proliferation in tissue culture: implications for axonal regeneration in the central nervous system.

A tissue culture model has been developed to examine the hypothesis that proliferating non-neuronal cells may constitute a physical and/or chemical barrier to regenerating neurons in the central nervous system. Explants from the sensorimotor cortex of 20-day-old fetal rats were cultured in serum medium (control) or serum medium containing 10(-5) M cytosine arabinoside (AraC), a mitotic inhibitor, for varying periods: 2-10, 4-12, 4-10, 4-8 and 4-7 days in vitro (DIV). The center and outgrowth zone of the explants were examined by phase-contrast microscopy at varying intervals between 3 and 18 DIV. The extent of central degeneration was greatest in explants treated with AraC from 2 DIV, and was least in the 4-7 day treated group in which only minimal degeneration was evident at 13 and 18 DIV. In the outgrowth zone at 18 DIV non-neuronal cell proliferation was controlled in the 4-10 day treated explants, although this was accompanied by extensive degeneration of neurites. Further examination of neurite viability, using a neurite viability ratio, revealed that degeneration was first evident at 6 DIV in the 2-10 day treated explants, but not until 9 or 13 DIV in any of the explants exposed to AraC from 4 days onwards. There was minimal degeneration in the 4-7 day treated explants. Electron microscopic examination revealed the presence of atypical inclusions in non-neuronal cells of 4-8 day treated explants, suggesting that the cytotoxic effect of AraC may be due to a disturbance in lipid and/or ganglioside metabolism. Quantitative electron microscopic analysis of the outgrowth zone at 18 DIV revealed a significant increase in the summated area of neuronal tissue (from 7 to 18 microns2/100 microns2) and a decline in the summated area of non-neuronal cells (from 83 to 61 microns2/100 microns2) for explants treated with AraC from 4 to 7 DIV compared to control. Diminishing the potential of non-neuronal cells to act as a barrier by controlling their proliferation may, therefore, be of importance in enhancing the regenerative response of central neurons.

Tissue culture analysis of neurite outgrowth in the presence and absence of serum: possible relevance for central nervous system regeneration.

A tissue culture model has been developed to examine the hypothesis that axons can only regenerate when their growing tips are surrounded by extracellular fluid containing proteins derived from the blood. Fetal rat cerebral explants were cultured in serum medium for 10 days, followed by serum-free (SF) medium (from which serum had been removed) until 18 days in vitro (DIV). All explants cultured in serum medium for 0-10 DIV exhibited greater than 77% neurite viability (neurite viability ratio, NVR, 3.10). This degree of neurite viability was maintained for those explants exposed to serum until 18 DIV (NVR 2.82 at 18 DIV). By contrast, explants maintained in SF medium from 10-18 DIV had a much lower NVR, which, by 18 DIV, had declined to 0.30 (7.5% viability). Transmission electron microscopic analysis of explants fixed at 18 DIV confirmed these phase-contrast results and also showed a predominance of axonal profiles within the neurite population. In the center of explants, tissue viability was in excess of 75% in both the serum and SF media, suggesting that serum is of primary importance for axonal extension rather than neuronal survival. These data strengthen the hypothesis that blood-derived proteins may be needed for prolonged regeneration.