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1.
J Endocr Soc ; 3(11): 2123-2134, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31687639

RESUMO

Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during in vitro fertilization.

2.
Elife ; 82019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31232694

RESUMO

The Mullerian ducts are the anlagen of the female reproductive tract, which regress in the male fetus in response to MIS. This process is driven by subluminal mesenchymal cells expressing Misr2, which trigger the regression of the adjacent Mullerian ductal epithelium. In females, these Misr2+ cells are retained, yet their contribution to the development of the uterus remains unknown. Here, we report that subluminal Misr2+ cells persist postnatally in the uterus of rodents, but recede by week 37 of gestation in humans. Using single-cell RNA sequencing, we demonstrate that ectopic postnatal MIS administration inhibits these cells and prevents the formation of endometrial stroma in rodents, suggesting a progenitor function. Exposure to MIS during the first six days of life, by inhibiting specification of the stroma, dysregulates paracrine signals necessary for uterine development, eventually resulting in apoptosis of the Misr2+ cells, uterine hypoplasia, and complete infertility in the adult female.

3.
Proc Natl Acad Sci U S A ; 114(9): E1688-E1697, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28137855

RESUMO

The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman's reproductive lifespan, resulting in menopause. Müllerian inhibiting substance (MIS) (or anti-Müllerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS-treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy.


Assuntos
Hormônio Antimülleriano/farmacologia , Antineoplásicos/efeitos adversos , Anticoncepcionais/farmacologia , Folículo Ovariano/efeitos dos fármacos , Reserva Ovariana/efeitos dos fármacos , Animais , Anticoncepção/métodos , Dependovirus/metabolismo , Feminino , Células da Granulosa/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência Ovariana Primária/prevenção & controle , Reprodução/efeitos dos fármacos
4.
J Neurosci Methods ; 212(1): 106-13, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23022696

RESUMO

The cholinergic neurons of the basal forebrain (BFCNs) in human and non-human primates are rich in the calcium binding protein calbindin-D(28k) (CB). We have shown a selective loss of CB from BFCNs in the course of normal aging, which appears to predispose these neurons to tangle formation and degeneration in Alzheimer's disease. Our previous preliminary investigation demonstrated that rodent BFCNs are devoid of CB. Here we confirm that rat choline acetyltransferase-rich BFCNs are devoid of CB immunoreactivity. We then describe a method for adeno-associated viral vector (AAV) induced expression of CB in rat BFCNs in vivo. We constructed AAV vectors bearing the CB gene under the control of the CMV promoter, or neuron-specific enolase (NSE) promoter, to bias expression in neurons. Both vectors resulted in CB expression in mouse neuronal cultures, and in rat brain following injections. AAV-NSE-CB resulted in more robust expression in neurons. Injections of 10 µl of AAV-NSE-CB in the BFCNs component located within the internal segment of globus pallidus and internal capsule resulted in expression of CB in 84% of BFCNs. Expression was optimum at 14 days. Injections of AAV-NSE-LacZ resulted in robust ß-galactosidase expression, but no CB immunoreactivity. Our results show that use of NSE promoter leads to high expression of genes in neurons and that the BFCNs can be targeted for expression of genes that are differentially expressed in the rodent and primate brains. These findings have important implications for gene replacement therapy in human BFCNs.


Assuntos
Neurônios Colinérgicos/metabolismo , Dependovirus/genética , Regulação da Expressão Gênica/genética , Prosencéfalo/citologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteína de Transporte de Acila/metabolismo , Animais , Calbindinas , Células Cultivadas , Colina O-Acetiltransferase/metabolismo , Vetores Genéticos/fisiologia , Masculino , Camundongos , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/genética , Especificidade da Espécie , Transdução Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
5.
J Neuropathol Exp Neurol ; 67(4): 309-18, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18379437

RESUMO

A large body of evidence indicates that basal forebrain cholinergic neurons are selectively vulnerable to degeneration early in Alzheimer disease (AD). Recent studies, however, demonstrate reductions in cortical activity of the cholinergic enzyme choline acetyltransferase only in late stages of AD. To address this apparent contradiction, we compared abnormalities in magnocellular basal forebrain cholinergic neurons and their axons in nondemented young (<65 years; n = 6), nondemented old (>65 years; n = 7), pathologically mild (n = 5), and pathologically severe (n = 5) AD cases. Cholinergic axon abnormalities (i.e. thickened fibers and ballooned terminals) were evident in nondemented middle-aged cases, increased in nondemented old cases, and reduced in density in severe AD. This suggests that loss of cortical cholinergic axons in AD occurs preferentially in fibers with these abnormalities. Paired helical filament 1-immunoreactive pretangles and tangles were observed as early as the third decade prior to their appearance in entorhinal/perirhinal cortex; they were increased in mild and severe AD. These results indicate that basal forebrain cholinergic neuron abnormalities are present very early in aging and in the course of AD. Therefore, despite the morphologic alterations, choline acetyltransferase activity, but not necessarily normal neuron functions, may be preserved.


Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Axônios/patologia , Colina O-Acetiltransferase/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Contagem de Células/métodos , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Grupo Polycomb , Mudanças Depois da Morte , Prosencéfalo/patologia , Fatores de Transcrição/metabolismo
6.
Exp Neurol ; 204(2): 640-57, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17274983

RESUMO

Unlike the development of acetylcholinesterase (AChE) activity, the postnatal development of the activity of the related enzyme butyrylcholinesterase (BuChE) in the rodent brain has not been investigated in a comprehensive manner. The purpose of the present study was to fill this gap. Development of histochemically visualized BuChE activity followed four distinct stages. Between birth and five postnatal days (P0-P5) BuChE staining of very low intensity was present in nearly all neurons in the forebrain and upper brainstem. Substantial BuChE activity was present in the endothelial cells of blood vessels and the cuboidal cells lining the ventricles. At P6-P10, BuChE neuronal staining of high to moderate intensity emerged in many areas, including certain thalamic nuclei (e.g. anterior group), a number of brainstem nuclei, and darkly stained neurons in the olfactory tubercle/piriform cortex. At P11-P17, the staining which emerged in earlier stages was darker and had expanded to include more neurons. A scattered population of BuChE-positive neurons of moderate to high intensity emerged in the neocortex and amygdala. Importantly, at P17, the very light staining present in all neurons since birth was no longer visible. At P18-P30, the number and staining intensity of cortical neurons displayed a gradual increase while the staining in certain thalamic nuclei was substantially decreased or completely disappeared (e.g. ventral lateral nucleus). A prominent feature of this stage was the emergence of BuChE activity in many fiber tracts. At P30, the adult pattern of staining was attained. The transient presence of BuChE activity of very low intensity in all neurons and of higher intensity in thalamic neurons supports the implied role for this enzyme in neuronal development.


Assuntos
Tronco Encefálico/enzimologia , Tronco Encefálico/crescimento & desenvolvimento , Butirilcolinesterase/metabolismo , Prosencéfalo/enzimologia , Prosencéfalo/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Tronco Encefálico/anatomia & histologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Prosencéfalo/anatomia & histologia , Ratos , Ratos Sprague-Dawley
7.
Brain Res ; 1060(1-2): 144-52, 2005 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-16212945

RESUMO

Acetylcholinesterase and butyrylcholinesterase activities emerge in association with plaques and tangles in Alzheimer's disease. These pathological cholinesterases, with altered properties, are suggested to participate in formation of plaques. The present experiment assessed the ability of rivastigmine, a clinically utilized agent that inhibits acetylcholinesterase and butyrylcholinesterase activities, to inhibit cholinesterases in plaques and tangles. Cortical sections from cases of Alzheimer's disease were processed using cholinesterase histochemistry in the presence or absence of rivastigmine. Optical densities of stained sections were utilized as a measure of inhibition. The potency of rivastigmine was compared with those of other specific inhibitors. Optimum staining for cholinesterases in neurons and axons was obtained at pH 8.0. Cholinesterases in plaques, tangles and glia were stained best at pH 6.8. Butyrylcholinesterase-positive plaques were more numerous than acetylcholinesterase-positive plaques. Rivastigmine inhibited acetylcholinesterase in all positive structures in a dose-dependent manner (10(-6)-10(-4) M). However, even at the highest concentration, faint activity remained. In contrast, rivastigmine resulted in complete inhibition of butyrylcholinesterase in all structures at 10(-5) M. Rivastigmine was equipotent to the specific acetylcholinesterase inhibitor BW284C51 and more potent than the butyrylcholinesterase inhibitors iso-OMPA and ethopropazine. In conclusion, rivastigmine is a potent inhibitor of acetylcholinesterase and a more potent inhibitor of butyrylcholinesterase in plaques and tangles. Unlike other cholinesterase inhibitors tested, rivastigmine inhibited cholinesterases in normal and pathological structures with the same potency. Thus, at the therapeutic concentrations used, rivastigmine is likely to result in inhibition of pathological cholinesterases, with the potential of interfering with the disease process.


Assuntos
Acetilcolinesterase/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Butirilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Fenilcarbamatos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/enzimologia , Placa Amiloide/efeitos dos fármacos , Placa Amiloide/enzimologia , Rivastigmina
8.
J Neuropathol Exp Neurol ; 62(6): 605-16, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12834105

RESUMO

Reports from our laboratory have indicated a substantial and specific loss of the calcium binding protein calbindin-D28K (CB) from the human basal forebrain cholinergic neurons (BFCN) in the course of normal aging. In the present set of experiments we determined the relationship between the age-related loss of CB and the presence and density of plaques and tangles in the brains of normal elderly. In 23 cases ranging in age from 20 to 93 years of age we observed plaques and tangles in the BFCN region and the cerebral cortex in a subset of cases. Plaques were seen in the basal forebrain in very few cases above 65 years. Plaque density in the basal forebrain and cortex displayed a significant negative correlation with the proportion of the BFCN, which contained CB immunoreactivity. However, the brains of 2 elderly cases that displayed a substantial loss of CB from the BFCN did not contain any plaques. Tangles were observed in the BFCN as early as 26 years of age. Only tangles in the entorhinal cortex showed a significant negative correlation with the loss of CB from the BFCN. It is likely that loss of CB from the BFCN and formation of plaques and tangles are part of general age-related processes that occur in parallel rather than being causally related.


Assuntos
Envelhecimento/metabolismo , Colina O-Acetiltransferase/metabolismo , Emaranhados Neurofibrilares/metabolismo , Neurônios/metabolismo , Placa Amiloide/metabolismo , Prosencéfalo/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Peptídeos beta-Amiloides/metabolismo , Calbindina 1 , Calbindinas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/patologia , Neurônios/patologia , Placa Amiloide/patologia , Prosencéfalo/patologia
9.
Exp Neurol ; 182(1): 220-31, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12821392

RESUMO

Calbindin-D(28k) (CB), calretinin (CRT), and parvalbumin (PV) are high-affinity cytosolic calcium (Ca(2+)) binding proteins (CBP) that have been found to regulate intracellular calcium concentrations in neurons through their buffering capacity and to protect neurons from insults that induce elevations of intracellular Ca(2+). In earlier studies we observed a substantial and neurochemically specific loss of CB from the human basal forebrain cholinergic neurons (BFCN) in the course of normal aging. In the present experiments we expanded our investigation of age-related changes in calcium binding proteins in the human brain by investigating the status of CB-, CRT-, and PV-positive neurons in 17 cortical areas. There was a trend toward a decrease in the number of CB-immunoreactive neurons in all areas studied. However, this trend reached significance in only 4 areas in which the loss of CB-positive neurons ranged between 20 and 46%. Immunoreactivity for CRT was also decreased in many areas and this difference reached significance in three regions (26-37%). Cortical neurons displaying PV immunoreactivity did not show an age-related change. Comparison with other neurochemically specific cortical neurons indicated a similar age-related loss of nonphosphorylated neurofilament and NADPH-d activity in only a few cortical areas. In contrast, neuronal acetylcholinesterase activity was increased in a few cortical areas. These observations indicate that loss of CBP-positive neurons occurs in restricted cortical regions and is not a specific change as other neurochemically specific neurons also display restricted age-related changes. Furthermore, the age-related changes in cortical CBP-positive neurons appear to be considerably smaller than similar changes in the BFCN. The age-related depletion of CBPs is likely to deprive neurons from the capacity to buffer intracellular calcium and thus to leave them vulnerable to pathological processes that can cause increased intracellular calcium and lead to their degeneration.


Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Parvalbuminas/biossíntese , Proteína G de Ligação ao Cálcio S100/biossíntese , Acetilcolinesterase/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/citologia , Calbindina 1 , Calbindina 2 , Calbindinas , Contagem de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , NADPH Desidrogenase/biossíntese , Fibras Nervosas Mielinizadas/metabolismo , Proteínas de Neurofilamentos/biossíntese , Neurônios/citologia
10.
J Comp Neurol ; 455(2): 249-59, 2003 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-12454989

RESUMO

Cholinergic neurons of the basal forebrain (BFCN) are selectively vulnerable in neurodegenerative disorders of the elderly, particularly in Alzheimer's disease (AD). We investigated age-related changes in the BFCN that may serve as a substrate for this vulnerability. We report a substantial and selective age-related loss of the calcium binding protein calbindin-D(28K) (CB) from the human BFCN. Unbiased stereological estimation indicated that, in individuals under age 65 years, 72% of the choline acetyltransferase (ChAT)-positive BFCN contained CB immunoreactivity. In individuals over age 65 years, only 28% of the BFCN contained CB immunoreactivity, a dramatic loss of 61%. Similar results were obtained using neuronal counts from matching single- or double-stained sections in a larger cohort. The loss of CB immunoreactivity was neurochemically specific. No age-related changes were observed in the number of ChAT- or low-affinity nerve growth factor receptor (p75(NTR))-immunoreactive profiles. The loss of CB was greatest in very old individuals, in whom a small loss of BFCN was observed. Furthermore, the loss of CB displayed the same pattern as the loss of BFCN in AD and was more substantial in the posterior compared with the anterior BFCN sector, suggesting a role for CB in the selective vulnerability of BFCN in AD. The depletion of CB from the BFCN is likely to deprive these neurons of the capacity to buffer high levels of intracellular Ca(2+) and thus to leave them vulnerable to pathological processes, such as those in neurodegenerative disorders, which can cause increased intracellular Ca(2+), thus leading to their degeneration.


Assuntos
Envelhecimento/metabolismo , Fibras Colinérgicas/metabolismo , Prosencéfalo/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Calbindina 1 , Calbindinas , Feminino , Humanos , Imuno-Histoquímica , Masculino
11.
Acta Neuropathol ; 103(1): 48-58, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11837747

RESUMO

The incidence, distribution and chemical composition of amyloid-beta (A beta) peptide-positive deposits were investigated in the lower primate species common marmoset (Callithrix jacchus). No A beta deposits were observed in the brains of 7 marmosets below 7 years of age. In 15 marmosets above 7 years, 60% displayed cortical A beta-immunoreactive plaques, 80% had A beta deposited in intracortical vessels and 87% displayed A beta deposits in meningeal vessels. The cerebral cortex of the oldest animal (15 years) contained a substantial density of deposits. A beta-immunoreactive plaques were found predominantly in association cortical zones followed by a lower density in paralimbic cortical areas. Deposits within vessels were most frequent in occipital cortex. A beta40 was found primarily in vascular deposits, while A beta42 was present in plaques. Approximately 20% of plaques and most vascular deposits displayed thioflavin S staining, indicative of the presence of fibrillar A beta. Varying proportions of A beta deposits contained acetylcholinesterase or butyrylcholinesterase activities and apolipoprotein E and alpha1-antichymotrypsin immunoreactivity. A few plaques contained immunoreactivity for amyloid precursor protein in swollen neurites. However, no abnormally phosphorylated tau immunoreactivity was present in these neurites. Survival analysis in a colony of marmosets indicated that only 6% of animals can be expected to survive beyond 7 years of age. These results indicate that the aged marmoset brain displays A beta deposits with a distribution and chemical composition similar to those found in the human. These similarities suggest that the aged marmoset may be a useful lower primate model for the study of the pathological effects of A beta. However, the relatively small number of animals which can be expected to reach old age severely limits the utility of this species as a model of A beta deposition.


Assuntos
Envelhecimento/patologia , Peptídeos beta-Amiloides/análise , Encefalopatias/patologia , Córtex Cerebral/patologia , Fragmentos de Peptídeos/análise , Animais , Apolipoproteínas E/análise , Benzotiazóis , Encefalopatias/epidemiologia , Encefalopatias/mortalidade , Callithrix , Córtex Cerebral/química , Córtex Cerebral/enzimologia , Colinesterases/análise , Modelos Animais de Doenças , Feminino , Incidência , Masculino , Neuritos/química , Neuritos/enzimologia , Neuritos/patologia , Placa Amiloide/química , Placa Amiloide/enzimologia , Placa Amiloide/patologia , Análise de Sobrevida , Tiazóis/análise , alfa 1-Antiquimotripsina/análise
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