Congenital Hypothyroidism is Associated With Impairment of the Leptin Signaling Pathway in the Hypothalamus in Male Wistar Animals in Adult Life.

The goal of this study is to investigate whether congenital hypothyroidism induced by MMI during gestation (G) or gestation plus lactation (GL) would affect the leptin action upon body weight control on hypothalamus. Six to eight pups per group were killed at 90 days of age. For statistical analysis one-way ANOVA followed by the Holm-Sìdak post hoc test was used. Hypothyroidism resulted in a significant increase in leptin serum levels in G 20% and GL 25% (p<0.04). There was a significant expression decrease of OBR in G 45% and GL 63%; pSTAT3 in G 56% and GL 51%; pERK in G 50% and GL 48%; POMC in G 41% and GL 46% (p<0.04), while a significant increase was assigned to SOCS3 in G 52% and GL 170% (p<0.04) protein expression. We can conclude that hypothyroxinemia condition in rats on adulthood results in impairment of the leptin signaling pathway via ObRb-STAT3 in the hypothalamus, which is likely to be involved in the leptin resistance.

Leptin and leptin receptor are expressed only in clear cells of rat epididymis epithelia / La leptina y el receptor de leptina se expresan solamente en células claras del epitelio epidídimario de rata

SUMMARY: Leptin is a 16 kilodaltons hormone secreted by adipose tissue and in the past few years it has been related to the reproductive system regulation. Leptin and its receptor (OBR) have been described in several reproductive organs and in different species but, in epididymis, there is still a lack of information. The aim of this work is to establish if leptin and its receptor are present on epididymis and where the production is occurring. At mRNA level the cauda portion showed a high expression of leptin (p<0.025) and OBRa (p<0.002) while at protein level the OBR expression was lower in cauda region (p<0.025) and leptin was not detected. The ratio between OBRa and OBRb was higher in both regions despite its total amount. By immunohistochemistry leptin and OBR were detected on epididymis epithelia, restricted to clear cells (CC). After efferent duct ligation (EDL) a decrease on leptin staining on CC was observed, suggesting that despite of epididymis production, most of leptin source may probably come from testis. Our results show that leptin and OBR, both mRNA and protein, are present on epididymis and exclusively in CC, suggesting that this tissue is responsive to the hormone and may have an important role on CC regulation.

RESUMEN: La leptina es una hormona de 16 kilodaltons secretada por el tejido adiposo y se ha relacionado en los últimos años con la regulación del sistema reproductivo. La leptina y su receptor (OBR) se han reportado en varios órganos reproductores y en diferentes especies sin embargo, en el epidídimo aún falta información. El objetivo de este trabajo fue establecer si la leptina y su receptor están presentes en el epidídimo y donde se produce. A nivel de ARNm la porción de cauda mostró una alta expresión de leptina (p <0,025) y OBRa (p <0,002) mientras que a nivel de proteína la expresión de OBR fue menor en la región de la cauda epididimaria (p <0,025) y no se detectó leptina. La relación entre OBRa y OBRb fue mayor en ambas regiones a pesar de su cantidad total. Por inmunohistoquímica se detectaron leptina y OBR en el epitelio del epidídimo restringido a células claras (CC). Después de la ligadura del conducto deferente (EDL) se observó una disminución en la tinción de leptina en CC, lo que sugiere que a pesar de la producción del epidídimo, la mayor parte de la fuente de leptina puede provenir probablemente del testículo. Nuestros resultados mostraron que la leptina y OBR, mRNA y proteína, están presentes en el epidídimo y exclusivamente en CC, lo que sugiere que este tejido es sensible a la hormona y puede tener un papel importante en la regulación CC.

The testis of the mice C57/BL6 offspring in adulthood have alterations due to maternal caffeine consumption.

PURPOSE: To investigate the effects of the maternal caffeine consumption during pregnancy to adult male testis mice offspring. METHODS: Twenty pregnant mice were divided into control group (c) and caffeine group (cf). dams received daily saline or 20 mg/kg of caffeine subcutaneously. Male offspring were monitored daily until 13th week. The testis were used to evaluate both the proliferation (pcna) and apoptosis (bax); leptin receptor (ob-r); aromatase; follicle stimulating hormone (fshr), luteinizing hormone (lhr) and androgen receptors (ar); steroidogenic acute regulatory protein (star); vascular endothelial growth factor (vegf) and estrogen receptors (erα and erß) by western blotting. Serum concentrations of testosterone, estradiol and leptin were measured. RESULTS: There was a significant reduction in food intake and the body mass gain (p<0.05) in the cf ; pcna (p=0.01), fshr (p=0.02), star (p=0.0007), vegf (p=0.009), ar (p=0.03) in the cf. while an increase were note in bax (p=0.01), ob-r (p=0.02), lhr (p=0.04) and in the aromatase (p=0.03) in the cf. only erα and erß were not changed by maternal caffeine. The serum testosterone levels in the cf offspring were 90% lower than in the c offspring (p=0.04). CONCLUSION: Maternal caffeine consumption has a role and alters the testis of the offspring in adulthood.

The testis of the mice C57/BL6 offspring in adulthood have alterations due to maternal caffeine consumption

To investigate the effects of the maternal caffeine consumption during pregnancy to adult male testis mice offspring. METHODS: Twenty pregnant mice were divided into control group (c) and caffeine group (cf). dams received daily saline or 20 mg/kg of caffeine subcutaneously. Male offspring were monitored daily until 13th week. The testis were used to evaluate both the proliferation (pcna) and apoptosis (bax); leptin receptor (ob-r); aromatase; follicle stimulating hormone (fshr), luteinizing hormone (lhr) and androgen receptors (ar); steroidogenic acute regulatory protein (star); vascular endothelial growth factor (vegf) and estrogen receptors (erα and erβ) by western blotting. Serum concentrations of testosterone, estradiol and leptin were measured. RESULTS: There was a significant reduction in food intake and the body mass gain (p<0.05) in the cf ; pcna (p=0.01), fshr (p=0.02), star (p=0.0007), vegf (p=0.009), ar (p=0.03) in the cf. while an increase were note in bax (p=0.01), ob-r (p=0.02), lhr (p=0.04) and in the aromatase (p=0.03) in the cf. only erα and erβ were not changed by maternal caffeine. The serum testosterone levels in the cf offspring were 90% lower than in the c offspring (p=0.04). CONCLUSION: Maternal caffeine consumption has a role and alters the testis of the offspring in adulthood.

Leptin regulates gonadotropins and steroid receptors in the rats ovary / La leptina regula las gonadotropinas y los receptores de esteroides en el ovario de las ratas

The leptin hormone is important to satiety and an important link between the nutritional status and reproductive processes. Owing to the contradictory effects of leptin on the ovary and the failure to clarify the precise mechanism by which leptin affects the ovary, our aim was to contribute to evaluation if leptin can directly regulate the gene expression of leptin itself and its receptors, and the expression of several genes related to the ovary function by a model of tissue culture. Ovaries from Wistar dams were used at 90 days of age and were submitted to medium with presence and absence of leptin. The results can demonstrate that leptin regulates gonadotropins and steroid receptors, which could suggest that the ovarian leptin role could be secondary to the changes in these receptorse expression in rats (AU)

La hormona leptina es importante en la sensación de la saciedad y un vínculo importante entre el estado nutricional y los procesos reproductivos. Debido a los efectos contradictorios de la leptina en el ovario y la falta de esclarecimiento del mecanismo exacto por el cual la leptina afecta el ovario, nuestro objetivo es contribuir a la evaluación si la leptina puede regular directamente la expresión del gen de la leptina sí mismo y sus receptores, y la expresión de varios genes relacionados con la función del ovario por un modelo de cultivo de tejidos. Los ovarios de las presas Wistar fueron usadas en los 90 días de edad y se sometieron a medio con presencia y ausencia de leptina. Los resultados pueden mostrar que la leptina regula las gonadotropinas y los receptores de esteroides, lo que podría sugerir que la función ovárica de la leptina podría ser secundario a los cambios en la expresión de sus receptores en ratas (AU)

Maternal malnutrition during lactation in Wistar rats: effects on elastic fibers of the extracellular matrix in the trachea of offspring.

OBJECTIVE: To investigate the effects of maternal protein malnutrition during lactation on the elastic fibers in the tracheas of Wistar rat pups. METHODS: At delivery, 12 male pups of two Wistar rat dams were equally divided into two groups: control, in which the dam received water and standard rat chow ad libitum during lactation; and protein-restricted (PR), in which the dam received water ad libitum and an isoenergetic PR diet (8% protein). At 21 days of age, the pups were killed and their tracheas were excised. The elastic fibers were stained with Weigert's resorcin-fuchsin (after oxidation) and evaluated under light microscopy. Morphometric determinations were performed by stereology, with the point-counting method, and expressed as volumetric densities. RESULTS: Elastic fibers, most having a longitudinal distribution, were identified beneath the tracheal mucosa. In addition, well-defined circular layers of elastic fibers were found around the inner and outer surfaces of the cartilaginous ring. There were no differences between the groups regarding the organization and distribution of the elastic fibers. The volumetric density of the elastic fibers of the pups in the control and PR groups was 2.46 ± 0.99% and 3.25 ± 1.13%, respectively (p < 0.01). CONCLUSIONS: The volumetric density of elastic fibers appears to be greater in rat pups breastfed by dams receiving a PR diet than in those breastfed by dams receiving a normal diet.

Desnutrição materna durante a lactação em ratos Wistar: efeitos sobre as fibras elásticas da matriz extracelular na traqueia dos filhotes / Maternal malnutrition during lactation in Wistar rats: effects on elastic fibers of the extracellular matrix in the trachea of offspring

OBJETIVO: Investigar os efeitos da desnutrição proteica materna durante a lactação sobre as fibras elásticas da traqueia de filhotes de ratos Wistar. MÉTODOS: Ao nascimento, 12 filhotes machos de duas ratas Wistar foram igualmente divididos em dois grupos: grupo controle, cuja mãe recebeu água e dieta padrão de laboratório ad libitum durante a lactação, e grupo restrição proteica (RP), cuja mãe recebeu água ad libitum e dieta isoenergética com RP (8% de proteína). Aos 21 dias de vida, os filhotes foram sacrificados, e suas traqueias foram ressecadas. As fibras elásticas foram coradas pelo método de resorcina-fucsina de Weigert (precedido de oxidação) e avaliadas sob microscopia óptica. As determinações morfométricas foram feitas por estereologia, utilizando o método de contagem de pontos, e expressas em densidade volumétrica. RESULTADOS: As fibras elásticas foram identificadas abaixo da mucosa traqueal, sendo a maioria em distribuição longitudinal. Além disso, camadas circulares bem definidas de fibras elásticas envolviam as superfícies interna e externa do anel cartilaginoso. Não houve diferenças entre os grupos quanto à organização e distribuição das fibras elásticas. A densidade volumétrica das fibras elásticas dos filhotes nos grupos controle e RP foi de, respectivamente, 2,46 ± 0,99% e 3,25 ± 1,13% (p < 0,01). CONCLUSÕES: Nossos resultados sugerem que a densidade volumétrica de fibras elásticas é maior em filhotes de ratos alimentados por fêmeas submetidas a dieta com RP do que naqueles de mães recebendo dieta normal.

OBJECTIVE: To investigate the effects of maternal protein malnutrition during lactation on the elastic fibers in the tracheas of Wistar rat pups. METHODS: At delivery, 12 male pups of two Wistar rat dams were equally divided into two groups: control, in which the dam received water and standard rat chow ad libitum during lactation; and protein-restricted (PR), in which the dam received water ad libitum and an isoenergetic PR diet (8% protein). At 21 days of age, the pups were killed and their tracheas were excised. The elastic fibers were stained with Weigert's resorcin-fuchsin (after oxidation) and evaluated under light microscopy. Morphometric determinations were performed by stereology, with the point-counting method, and expressed as volumetric densities. RESULTS: Elastic fibers, most having a longitudinal distribution, were identified beneath the tracheal mucosa. In addition, well-defined circular layers of elastic fibers were found around the inner and outer surfaces of the cartilaginous ring. There were no differences between the groups regarding the organization and distribution of the elastic fibers. The volumetric density of the elastic fibers of the pups in the control and PR groups was 2.46 ± 0.99% and 3.25 ± 1.13%, respectively (p < 0.01). CONCLUSIONS: The volumetric density of elastic fibers appears to be greater in rat pups breastfed by dams receiving a PR diet than in those breastfed by dams receiving a normal diet.

Maternal malnutrition during lactation alters gonadotropin-releasing hormone expression in the hypothalamus of weaned male rat pups.

Gonadotropin-releasing hormone (GnRH) is the key hormone regulating reproduction. Its feedback regulation is exercised by estradiol. The early postnatal period is critical for sexual differentiation. Despite the fact that malnutrition-related reproductive suppression in rats is a well-documented phenomenon, we had no knowledge, until now, on how maternal malnutrition affects GnRH expression and estradiol serum concentrations of weaned pups. Six pregnant Wistar rats were separated into three groups at delivery with 6 pups each: control group (C) with free access to a standard diet containing 23% protein; protein energy restricted group (PER) with free access to an isoenergy and 8% protein diet; and an energy-restricted (ER) group receiving a standard diet in restricted quantities, which were calculated according to the mean ingestion of the PER group. At 21 days post partum, the animals were killed and the serum estradiol was evaluated by radioimmunoassay. Immunohistochemistry for GNRH was performed. The serum estradiol concentration was decreased in PER and ER groups compared with C (PER, 34%; ER, 19%;P < 0.01) and the staining of GNRH was restricted to arcuate nucleus and median eminence in the control group while in PER and ER stained processes aligned with the third ventricle wall (periventricular nucleus) were present. In conclusion, our data reinforce the concept that the maternal nutritional state during lactation is critical for sexual maturation since maternal malnutrition resulted in a neuron migration delay evidenced by an altered GnRH expression profile, probably a consequence of low estradiol serum levels.

Metabolic programming of lipid profile and reproductive organs weight by leptin treatment on early life.

PURPOSE: To evaluate whether the neonatal leptin treatment during the first days of life can program the male reproductive organs weight and the lipid profile. METHODS: At birth 6 dams were divided into 2 groups: Leptin - each pup was injected with 50microL of recombinant rat leptin (80ng/g BW, sc), for the first 10 d of lactation; Control - each pup received the same volume of saline. After weaning, all pups received unlimited access to food until 190 days of age when they were killed. Values are given as mean + or - SEM of 6 animals and Test t Student was used to analyze the results. RESULTS: The leptin treatment resulted in a significant increase in body weight (Control= 411.8 + or - 16.31; Leptin= 481.8 + or - 11.29, p=0.005) and food consumption (Control= 25.32 + or - 0.09; Leptin= 32.42 + or - 0.15, p=0.0001) and a significant reduction in triglycerides levels (Control= 540.0 + or - 117.9; Leptin= 93.25 + or - 15.21, p=0.006) and in the weight of hypothalamus (Control= 0.234 + or - 0.016; Leptin= 0.154 + or - 0.015, p=0.007), pituitary (Control= 0.104 + or - 0.0120; Leptin= 0.033 + or - 0.012, p=0.003), testis (Control= 3.75 + or - 0.055; Leptin= 3.19 + or - 0.10, p=0.002) and prostate (Control=1.641 + or - 0.1389; Leptin= 0.91 + or - 0.07, p=0.001). CONCLUSION: Leptin treatment on the first days of life can program the reproductive organs weight and the lipid profile of the progeny.

Metabolic programming of lipid profile and reproductive organs weight by leptin treatment on early life / Programação metabólica do perfil lipídico e peso dos órgãos do sistema reprodutor pelo tratamento com leptina nos primeiros dias de vida

PURPOSE: To evaluate whether the neonatal leptin treatment during the first days of life can program the male reproductive organs weight and the lipid profile. METHODS: At birth 6 dams were divided into 2 groups: Leptin - each pup was injected with 50μL of recombinant rat leptin (80ng/g BW, sc), for the first 10 d of lactation; Control - each pup received the same volume of saline. After weaning, all pups received unlimited access to food until 190 days of age when they were killed. Values are given as mean ± SEM of 6 animals and Test t Student was used to analyze the results. RESULTS: The leptin treatment resulted in a significant increase in body weight (Control= 411.8±16.31; Leptin= 481.8±11.29, p=0.005) and food consumption (Control= 25.32±0.09; Leptin= 32.42±0.15, p=0.0001) and a significant reduction in triglycerides levels (Control= 540.0±117.9; Leptin= 93.25±15.21, p=0.006) and in the weight of hypothalamus (Control= 0.234±0.016; Leptin= 0.154±0.015, p=0.007), pituitary (Control= 0.104±0.0120; Leptin= 0.033±0.012, p=0.003), testis (Control= 3.75±0.055; Leptin= 3.19±0.10, p=0.002) and prostate (Control=1.641±0.1389; Leptin= 0.91±0.07, p=0.001). CONCLUSION: Leptin treatment on the first days of life can program the reproductive organs weight and the lipid profile of the progeny.

OBJETIVO: Avaliar se o tratamento neonatal com leptina durante os primeiros dias de vida poderia programar o peso dos orgãos do sistema reprodutor masculino e o perfil lipídico. MÉTODOS: Ao nascimento seis ratas-mãe foram distribuídas em dois grupos: Leptina - cada filhote recebeu 50μL de leptina recombinante (80ng/gPC, SC) nos primeiros 10 dias de lactação; Controle - cada filhote recebeu o mesmo volume de salina. Após o desmame, todos os filhotes tiveram acesso ilimitado a ração até 190 dias de idade quando foram mortos. Os dados são expressos como média ± erro padrão e foram analisados pelo teste t Student. RESULTADOS: O tratamento com leptina resultou em aumento significativo no peso corporal (Control= 411.8±16.31; Leptin= 481.8±11.29, p=0.005) e consumo alimentar (Control= 25.32±0.09; Leptin= 32.42±0.15, p=0.0001) e redução significativa nos níveis séricos de triglicerídeos (Control= 540.0±117.9; Leptin= 93.25±15.21, p=0.006), no peso do hipotálamo (Control= 0.234±0.016; Leptin= 0.154±0.015, p=0.007), hipófise (Control= 0.104±0.0120; Leptin= 0.033±0.012, p=0.003), testículo (Control= 3.75±0.055; Leptin= 3.19±0.10, p=0.002) e próstata (Control=1.641±0.1389; Leptin= 0.91±0.07, p=0.001). CONCLUSÃO: O tratamento com leptina nos primeiros dias de vida pode programar o peso dos órgãos do sistema reprodutor e o perfil lipídico da prole.

Maternal malnutrition during lactation alters the folliculogenesis and gonadotropins and estrogen isoforms ovarian receptors in the offspring at puberty.

In this study, we aimed to evaluate whether maternal malnutrition during lactation alters the folliculogenesis and the expression of the gonadotropins and estrogen isoforms ovarian receptors in the offspring at puberty. At parturition, dams were randomly assigned to the following groups: control (C) group, with free access to a standard laboratory diet containing 23% protein and protein-energy-restricted (PER) group, with free access to an isoenergy and protein-restricted diet containing 8% protein. After weaning, the female pups had free access to standard laboratory diet. The maternal malnutrition caused a significant increase in the number of preantral (C=13.72+/-2.87; PER=26.36+/-3.03, P<0.01) and small antral follicles (C=9.32+/-1.35; PER=17.64+/-2.33, P<0.01) and decrease in the number of primordial (C=11.72+/-1.37; PER=3.92+/-0.60, P<0.01) and Graafian follicles (C=1.84+/-0.21; PER=0.96+/-0.11, P<0.01), and corpus luteum (C=2.00+/-0.28; PER=0.80+/-0.31, P<0.01). The estradiol serum concentration was significantly higher (C=67.86+/-4.39; PER=83.29+/-2.68, P<0.05) while testosterone serum concentration did not show statistical difference (C=0.09+/-0.02; PER=0.11+/-0.01, P>0.05) in the PER group. In relation to the receptors expression, maternal malnutrition led to a significant increase in the amount of Fshr (C=0.89+/-0.04; PER=1.07+/-0.03, P<0.05) and Lhcqr (C=0.87+/-0.15; PER=1.33+/-0.08, P<0.05) transcripts and a significant decrease in the amount of Ar (C=0.59+/-0.006; PER=0.13+/-0.080, P<0.05), ER alpha (Esr1) (C=3.33+/-0.71; PER=0.74+/-0.50, P<0.05), ER beta 1 (Esr2) (C=1.33+/-0.06; PER=0.49+/-0.36, P<0.05), and ER beta 2 (Esr2) (C=3.28+/-0.60; PER=0.62+/-0.34, P<0.05) transcripts. In conclusion, perinatal maternal malnutrition can directly affect folliculogenesis at puberty probably as a consequence of changes in the ovarian expression of gonadotropins, androgen and estrogens isoforms receptors. Long-term sexual alterations could be expected in this experimental model, since a reduction in the primordial follicle number is observed, which can result in a decrease in the reproductive lifetime and an earlier termination of breeding capacity.

Stereological evaluation of the seminiferous tubules of rats after maternal undernutrition during the lactation period.

The goal of this study is to evaluate, through stereological methods, some structural aspects of offspring testes whose dams were submitted to protein and energy-restricted diets during the lactation period. At birth, dams were separated into 3 groups: control group (C), receiving a diet with 23% protein; protein-restricted group (PR), receiving a diet with 8% protein; energy-restricted group (ER), receiving a diet with 23% protein in restricted quantities. At weaning, the offspring was anesthetized and perfused with formalin solution. Then, the testes were excised and processed using routine histological methods. Compared to the C group, both PR and ER groups had a significant reduction in the testis weight (PR = 65%, ER = 60%, p < 0.01), in the total area (PR = 23%, ER = 32%, p < 0.01), in the luminal area (PR = 30%, ER = 36%, p < 0.01), in the epithelial area (PR = 21%, ER = 27%, p < 0.01), and in the epithelial height (PR = 17%, ER = 23%, p < 0.01) of the seminiferous tubule. We conclude that maternal malnutrition during lactation leads to structural changes in the testis that could be responsible for future alterations in this organ physiology.

Comportamento alimentar de ratos adultos submetidos à restrição protéica cujas mães sofreram desnutrição durante a lactação / Feeding behavior of adult rats submitted to protein malnutrition whose members received protein restricted diets during lactation

Foi avaliado o consumo alimentar de animais adultos submetidos à restrição protéica, cujas mães receberam dieta hipoprotéica ou hipocalórica na lactação: controle (C) ração normal com 23 por cento de proteína; restrição protéica (RP) 8 por cento de proteína; restrição energética (RE) 23 por cento de proteína, em quantidade restrita à ingerida pelo grupo restrição protéica. Após o desmame todos os filhotes receberam ração normal até 60 dias e nesta época foram submetidos, por 21 dias, ao seguinte tratamento: (C/C)-filhotes de mães C recebendo ração normal; (restrição protéica/controle)-filhotes de mães Controle, recebendo ração hipoprotéica; (C/RP)-filhotes de mães RP recebendo ração normal; (RP/RP)-filhotes de mães RP recebendo ração hipoprotéica; (C/RE)-filhotes de mães RE recebendo ração normal; (RP/RE)-filhotes de mães RE recebendo ração hipoprotéica. Os filhotes de mães RP consumiram menos ração até 57 dias (p<0,01), enquanto os filhotes de mães RE normalizaram a ingestão aos 37 dias. Aos 81 dias, os animais submetidos à restrição protéica consumiram menos ração (p<0,01). A dieta materna na lactação modificou o consumo alimentar e o peso corporal da prole na vida adulta, estando, possivelmente, a restrição protéica associada a uma alteração permanente no controle hipotalâmico da seleção de nutrientes da prole