Electroacupuncture intervention increases testosterone level of aged rats by activating ERK/Nrf2/HO-1 signaling of Leydig cells / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on anti-oxidant function of Leydig cells in aged rats with low testosterone, so as to reveal its underlying mechanism of anti-aging of male reproduction. METHODS: Eighteen 20 months old SD rats were divided into aged control, medication and EA groups(n=6 in each group), and other 6 young male SD rats (2 months of age) were used as the youth control group. The rats of the youth and the aged control groups received subcutaneous injection of 0.9% normal saline (7 mg·kg-1·3 d-1) for 8 weeks, and those of the medication group received abdominal subcutaneous injection of Testosterone Propionate (7 mg·kg-1·3 d-1) for 8 weeks. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Shenshu" (BL23) and "Guanyuan" (CV4) for 15 min, once daily for 8 weeks except the weekends. The levels of serum total testosterone (TT) and free testosterone (FT) were determined by enzyme linked immune sorbent assay (ELISA), the immunoactivity of phosphorylated extracellular signal-regulated protein kinase (p-ERK) was detected by immunohistochemistry, and the expression levels of ERK, p-ERK, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins in the testis tissues were determined by Western blot. RESULTS: Before and after the treatment, the levels of serum TT and FT in the aged control group were significantly lower than those of the youth control group (P0.05). CONCLUSION: EA intervention may increase testosterone level of the aged rats, which may be related to its effects in triggering ERK /Nrf2 /HO-1 signaling (anti-oxidative stress signal pathway) in the testis.

Electroacupuncture Intervention Improves Physical Power of Aged Rats with Low Testosterone by Reducing Chronic Inflammatory Response of Leydig Cells / 针刺研究

OBJECTIVE: To assess the effect of electroacupuncture (EA) on chronic inflammatory response of Leydig cells in aged rats with low testosterone, so as to investigate its underlying mechanism of anti-male reproductive aging. METHODS: Twenty-four 20 months old SD rats were randomly divided into EA, medication and aged control groups (n＝ 8 in each), and other 8 young SD rats (2 months of age) were used as the youth control group. EA (2 Hz/100 Hz, 1 mA) was applied to "Guanyuan"(CV 4) and bilate-ral "Shenshu"(BL 23) for 15 min, once daily for 8 weeks except the weekends. The medication group received abdominal subcutaneous injection of testosterone propionate (7 mg• kg－1• 3 d－1) for 8 weeks. The aged control group and the youth control group received subcutaneous injection of 0.9% normal saline, with the same dose and same treatment frequency as those of the medication group. The rats' physical power was assessed according to the exhausted swimming duration, and the levels of serum total testosterone (TT) and free testosterone(FT) were determined by ELISA. The pathological changes of the testis tissue were detected by using H．E.staining, and the immunoactivity of cyclooxygenase-2 (COX-2) in Leydig cells was detected by immunohisto-chemistry. The expression levels of nuclear factor-κB p 65 (NF-κB p 65), COX-2, interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) proteins in the testis tissues were determined by Western blot. RESULTS: Before and after treatment, the exhaustive swimming duration and the levels of serum TT and FT in the aged control group were significantly lower than those of the youth control group (P < 0.01). After the treatment, the exhaustive swimming duration and serum TT and FT in the EA and medication groups were notably higher than those in the aged control group (P<0.01). HE staining showed that the incompleteness of basement membrane of spermatogenic tubules, reduction of spermatogenic cells and supporting cells and irregularity of Leydig cells in the testis tissue of the aged rats were relatively milder after EA intervention. Compared with the youth control group, the expression levels of NF-κB p 65 and COX-2, IL-1 β and TNF-α in the testicular tissue were significantly higher in the aged control group (P<0.01),while in compared with the aged control group, the expression levels of NF-κB p 65, COX-2, IL-1 β and TNF-α proteins were significantly down-regulated in the EA group (P<0.01). CONCLUSION: EA intervention can improve the physical power of the aged rats with low testosterone, which may be related to its effects in up-regulating TT and FT levels, and in reducing chronic inflammatory response in the testis tissue.

Atuação das proteínas do relógio na senescência reprodutiva de ratas Wistar / Clock protein action in the senescence reproductive of female Wistar rats

O envelhecimento é considerado processo multidimensional no qual fatores ambientais podem proteger ou, inversamente, agravar seus sinais, de maneira não linear, nos processos fisiológicos e neurocomportamentais. Durante este processo, os ritmos circadianos são interrompidos ou fragmentados com dissociação consequente dos ritmos circadianos do indivíduo e disfunções relacionadas ao relógio circadiano contribuem para o envelhecimento e para patologias a ele relacionadas. O objetivo deste estudo foi averiguar possível alteração temporal do sistema CLOCK no eixo HPG e a relação com às alterações hormonais que caracterizam a periestropausa. Foram utilizadas fêmeas adultas com ciclo estral regular (CD) na fase do diestro e fêmeas senis com ciclo estral irregular e persistência da fase do diestro (IDP). Para análises de expressão gênica dos clock genes Per2, Rev-erbα e Bmal1 no eixo HPG, foram utilizados punchs das regiões do NSQ, onde também foi analisado RNAm de AVP, APO e HMB destes animais, além da adenohipófise e ovários dos quais se extraiu o RNA para confecção do cDNA e realização de qPCR. A determinação da atividade neuronal vasopressinérgica no NSQ foi realizada por imunoistoquíca com dupla marcação para cFos e AVP em tecido previamente fixado com paraformaldeído. A concentração plasmática de gonadotrofinas foi determinada por radioimunoensaio. De modo geral, os animais IDP revelaram alterações no perfil de expressão gênica durante o fotoperíodo, com redução de amplitude, deslocamento/desalinhamento de fase e ausência de antifase. O NSQ de animais IDP apresentou menor expressão de Rev-erbα e maior expressão de RNAm para AVP em relação ao grupo CD. A quantificação relativa de Bmal1 foi semelhante em ambos os grupos e não houve diferenças entre grupos na expressão de Per2. Na APO, animais IDP apresentaram maior expressão de Per2 e menor quantidade de RNAm para Rev-erbα. No HMB observou-se menor expressão para Per2 e Rev-erbα e maior expressão de Bmal1 nas fêmeas IDP. Per2 e Bmal1 na adenohipófise tiveram menor expressão que o gene Rev-erbα no grupo senil e o ovário destes animais revelou maior expressão para Per2 e Rev-erbα, em comparação com os animais CD. As concentrações plasmáticas de FSH foram maiores nas fêmeas com ciclo irregular (2,05 ± 0,44 ng/mL), principalmente durante a fase clara, assim como o LH (0,24 ± 0,07 ng/mL), cujos maiores valores foram encontrados durante a fase escura e com perfil semelhante ao RNAm de AVP. As imunomarcações revelaram alta atividade vasopressinérgica na porção dorsomedial do NSQ das fêmeas IDP. Juntos estes dados permitem concluir que há desarranjo na expressão temporal dos genes Per2, Rev-erbα, Bmal1 que compõem a maquinaria molecular do relógio circadiano, bem como de RNAm para AVP no NSQ, de fêmeas Wistar na periestropausa. Além disso, a maior atividade neuronal vasopressinérgica e a ausência de oscilação de Rev-erbα e Bmal1 no NSQ destes animais, comprometem a correta comunicação do relógio central do NSQ com o eixo HPG, inviabilizando a manutenção da fertilidade feminina e contribuindo para a senescência reprodutiva(AU)

Aging is considered a multidimensional process in which environmental factors can protect or, conversely, aggravate its signals, non-linearly, in physiological and neurobehavioral processes. During this process, circadian rhythms are disrupted or fragmented with consequent dissociation of the individual's circadian rhythms and circadian clock-related dysfunctions contribute to aging and related pathologies. The objective of this study was to investigate possible temporal alteration of the CLOCK system in the HPG axis and the relation with the hormonal changes that characterize periestropause. Adult females with regular estrus cycle in the diestrous phase (RD) and old females with irregular estrous cycle and persistent diestrous phase (IPD). For analyzes of the gene expression of the genes Per2, Rev-erbα and Bmal1 in the HPG axis, punchs from the NSQ regions were used, where AVP, POA and MBH RNAm from these animals were also analyzed, as well as the adenohypophysis and ovaries from which they were extracted the RNA for cDNA production and qPCR performance. The determination of the vasopressinergic neuronal activity in the NSQ was performed by immunohistochemical with double labeling for cFos/AVP in tissue previously fixed with paraformaldehyde. The plasma concentration of gonadotrophins was determined by radioimmunoassay. In general, the IPD animals show alterations in the gene expression profile during the period analyzed, with amplitude reduction, phase shift / misalignment and absence of antiphase. The NSQ of IPD animals presented lower expression of Rev-erbα and higher RNAm expression for AVP than RD group. The relative quantification of Bmal1 was similar in both groups and there were no differences between groups in the expression of Per2. In PAO, IPD animals showed higher expression of Per2 and less amount of RNAm for Rev-erbα. MBH showed lower expression for Per2 and Rev-erbα and higher Bmal1 expression in IPD females. Per2 and Bmal1 in the adenohypophysis had lower expression than the Rev-erbα gene in the old group and the ovary of these animals showed higher expression for Per2 and Rev-erbα, in related to to the RD animals. Plasma concentrations of FSH were higher in females with irregular cycle (2.05 ± 0.44 ng / mL), mainly during the light phase, as well as LH (0.24 ± 0.07 ng / mL) whose values were found during the dark phase and with a profile similar to AVP RNAm. Immunolabeling demonstrated high vasopressinergic activity in the dorsomedial portion of the NSQ of the IPD females. Together these data allow us to conclude that there is a breakdown in the temporal expression of the Per2, Rev-erbα, Bmal1 genes that make up the molecular machinery of the circadian clock, as well as RNAm for AVP in NSQ of Wistar females in peri-masterpause. In addition, the increased vasopressinergic neuronal activity and the absence of Rev-erbα and Bmal1 oscillation in the NSQ of these animals compromise the correct communication of the central clock of the NSQ with the HPG axis, making it impossible to maintain female fertility and contributing to reproductive senescence(AU)

Avaliação da reserva ovariana na propedêutica da infertilidade / Ovarian reserve evaluation in the propedeutic of infertility

A atenção dada ao potencial reprodutivo diante o envelhecimento ovariano foi realçada pela tendência da mulher moderna de postergar a maternidade e pela procura cada vez maior por serviços de reprodução assistida. Nesse contexto, a avaliação da reserva folicular ovariana surge como tentativa de aferir sua capacidade funcional, melhor aconselhar os casais interessados e nortear protocolos de estimulação, para amenizar encargos emocionais e financeiros de um processo cujos resultados ainda se encontram aquém do desejado. Neste texto, abordam-se a situação atual de marcadores endócrinos basais e dinâmicos e os marcadores ecográficos comuns na avaliação da reserva ovariana em mulheres candidatas a ciclos assistidos.

The attention given to reproductive potential along ovarian ageing process has been enhanced with the trends of modern women to postpone maternity and due to progressive number of couples looking for assisted reproduction treatment. In this context, evaluation of ovarian follicular reserve has been taken into account, with the aim to determine gonadal functional capacity, to afford a better counselling of couples and to coordinate stimulation protocols, diminishing emotional and financial onuses of a complex process with still unsatisfactory results. The scope of this article is to briefly present current situation of basal and dynamic endocrine, and sonographic markers commonly used in ovarian reserve evaluation in candidates for assisted reproductive programs.

Analysis of Serum Levels of Anti-Mullerian Hormone, Inhibin B, Insulin-Like Growth Factor-I, Insulin-Like Growth Factor Binding Protein-3, and Follicle-Stimulating Hormone with Respect to Age and Menopausal Status

This study was undertaken to investigate age-dependent and postmenopausal changes in the serum levels of anti-Mullerian hormone (AMH), inhibin B, insulin-like growth factor (IGF)-I, IGF-binding protein-3 (IGFBP-3), and follicle-stimulating hormone (FSH), and to determine which of these markers best reflects the aging process in women. A total of 144 women aged 20-59 yr were enrolled in this cross-sectional study. Blood samples were obtained on cycle day 3 of regularly menstruating women (n=111), or at random in postmenopausal women (n=33). Data were analyzed with respect to premenopausal women age groups and compared in pre- and postmenopausal women. Area under the receiver operating characteristic curve (ROCAUC) analyses were performed to assess the ability of each marker to discriminate between the pre- and postmenopausal status. Serum levels of AMH, IGF-I, and IGFBP-3 decreased and serum levels of FSH increased significantly with age in premenopausal women. Serum luteinizing hormone (LH) was higher and inhibin B was lower in women in their 20-30's than in 40's. Serum levels of AMH and IGF-I showed a consistent decrease with all age groups. ROCAUC analysis showed that the diagnostic accuracy of AMH for menopausal status was similar to those of FSH, LH, and inhibin B, and was better than that of IGF-I. In conclusion, the serum AMH level appears to be the best marker of the aging process in premenopausal women.