Role of amygdala kisspeptin in pubertal timing in female rats

PLoS ONE, Volume 12, No. 8, Year 2017

Interprétation

To investigate the mechanism by which maternal obesity disrupts reproductive function in offspring, we examined Kiss1 expression in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei, and posterodorsal medial amygdala (MePD) of pre-pubertal and young adult offspring. Sprague-Dawley rats were fed either a standard or energy-dense diet for six weeks prior to mating and throughout pregnancy and lactation. Male and female offspring were weaned onto normal diet on postnatal day (pnd) 21. Brains were collected on pnd 30 or 100 for qRT-PCR to determine Kiss1 mRNA levels. Maternal obesity increased Kiss1 mRNA expression in the MePD of pre-pubertal male and female offspring, whereas Kiss1 expression was not affected in the ARC or AVPV at this age. Maternal obesity reduced Kiss1 expression in all three brain regions of 3 month old female offspring, but only in MePD of males. The role of MePD kisspeptin on puberty, estrous cyclicity and preovulatory LH surges was assessed directly in a separate group of post-weanling and young adult female rats exposed to a normal diet throughout their life course. Bilateral intra-MePD cannulae connected to osmotic mini-pumps for delivery of kisspeptin receptor antagonist (Peptide 234 for 14 days) were chronically implanted on pnd 21 or 100. Antagonism of MePD kisspeptin delayed puberty onset, disrupted estrous cyclicity and reduced the incidence of LH surges. These data show that the MePD plays a key role in pubertal timing and ovulation and that maternal obesity may act via amygdala kisspeptin signaling to influence reproductive function in the offspring. All procedures were conducted in accordance with the United Kingdom Home Office Animals (Scientific Procedures) Act 1986. The protocols were approved by the Committee on the Ethics of Animal Experimentation of King’s College London. Adult female Sprague-Dawley rats obtained from Charles River (Margate, UK) were used as breeders in our facility at King’s College London; producing animals for study 1 and 2. A separate group of adult female Sprague-Dawley rats (Charles River) fed a normal diet throughout their life course was used in study 3. The rats were housed under controlled conditions (12 h light, 12 h dark cycle, lights on at 0700 h; temperature 22°C ± 2°C) with ad libitum access to food and water. Female Sprague-Dawley rats were fed either a standard chow diet (RM3, Special Dietary Services, Essex, UK) or a highly palatable energy-dense obesogenic diet consisting of 20% animal lard, 10% simple sugars, 28% polysaccharide, and 23% protein (w/w); energy 4.5 kcal/g (Special Dietary Services) and supplemented ad libitum with sweetened condensed milk [∼55% simple sugars and 8% fat, 8% protein (w/w); Nestlé] and fortified with mineral and vitamin mix (AIN 93G; Special Diets Services). The animals were maintained on these diets for 6 weeks before mating, during pregnancy and lactation. The effects of these diets on maternal phenotype has been described previously [25]. Litter size was standardised to 8 pups (4 male, 4 female) 48 hours after birth. All offspring were weaned at postnatal day (pnd) 21 and subsequently fed RM1 diet ad libitum. In subsequent experimental groups, 2 males and 2 females from each litter were used. Male and female offspring of control (OffCon) and obese (OffOb) dams were culled on pnd 30 (OffCon, n = 8 per sex; OffOb, n = 10 per sex) or 100 (OffCon, n = 8 per sex; OffOb, n = 10 per sex) for determination of Kiss1 mRNA expression in ARC, AVPV and MePD. These offspring were prepubertal and without significant difference in body weight at pnd 30 as described previously [25]. Animals were killed by decapitation and brains rapidly removed, snap frozen on dry ice and stored at -80°C until processing. Brains were cut into 300μm thick coronal sections using a cryostat (Bright Ltd., Cambridgeshire, UK) and mounted on coated polysine slides (ThermoFisher Scientific, Braunschweig, Germany). Brain punches were taken using the micropunch method [26,27] with coordinates obtained from the rat brain atlas of Paxinos and Watson [28]. For both ARC and AVPV nuclei, a single midline punch (1 mm diameter) was taken from bregma -1.7 to 3.9 and 0.26 to -1.3 respectively, while bilateral punches (0.6 mm diameter) from the MePD were taken from bregma -2.2 to -3.6. The punched sections were fixed with formalin and stained with crystal violet to confirm correct punch positioning under a microscope. Total RNA was extracted from the punched ARC, AVPV and MePD tissues for each rat using TRI reagent (Sigma-Aldrich, Poole, UK) and reverse transcribed using the reverse transcriptase Superscript II (Invitrogen, Carlsbad, CA, USA) and random primer following the manufacturer’s instructions. Hypoxanthine phosphoribosyltransferase 1 (Hprt1) mRNA was used as a reference gene for normalization of target gene. The primers used for quantitative real-time PCR are shown in Table 1. The primer pairs selected were designed to amplify across at least one intron, ruling out the possibility of identical size bands resulting from genomic DNA amplification. Reaction conditions for Kiss1 were optimized to give the best results for the primer and for the different quantities of target in samples [29]. The sample cDNA prepared as above was used as a template for the PCR using the Applied Biosystems® ViiA™ 7 Real-Time PCR System. During PCR, the amplified cDNA products were detected after each annealing phase in real time using the QuantiTect fast SYBR Green kit (QIAGEN, Hilden, Germany). Each reaction included 2 μl sample cDNA (optimized so that sample values of the PCR product were within the standard curve), 0.8 μl each of 10 μM antisense and sense primers, 4 μl QuantiTect SYBR Green mix, and 0.4 μl water to give a total reaction volume of 8 μl. The PCR cycling conditions for Kiss1 were initial denaturation and activation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 sec and annealing at 56°C for 10 sec and 72°C for 10 sec. The Hprt1 reaction conditions were 5 min at 95°C for one cycle, then 10 sec at 95°C, 10 sec at 55°C, and 10 sec at 72°C for 40 cycles. Expression level of Kiss1 was determined by the threshold cycle (Ct) value in the exponential phase of the PCR reaction and normalized to the respective Hprt1 Ct value using the comparative Ct method [30]. Post-weanling female Sprague-Dawley rats fed a normal diet prior to pregnancy and during pregnancy and lactation were used to determine the effect of MePD kisspeptin antagonism on pubertal timing. All surgical procedures were carried out under a combination of ketamine (Vetalar, 600 mg/kg, i.p.; Pfizer, Sandwich, UK) and xylazine (Rompun, 60 mg/kg, i.p.; Bayer, Newbury, UK) anesthesia. On pnd 21, animals were anaesthetized and secured in a David Kopf stereotaxic frame and small holes drilled into the skull at a location above the MePD after a small incision of the scalp. A 28-gauge cannula (Plastics One, Roanoke, VA, USA) was fitted bilaterally towards the MePD. The stereotaxic coordinates for implantation of the cannulae previously optimized [24] were 2.5 mm posterior to bregma (AP), 3.2 mm lateral (ML), and 7.8 mm below the surface of the dura (DV) using the rat brain atlas of Paxinos and Watson [28]. An osmotic mini-pump (Model 2002; Alza Corp, Mountain View, CA, USA) prefilled with kisspeptin receptor antagonist (Peptide 234; Sigma Adrich) or artificial cerebrospinal fluid (aCSF) was attached to the cannula with silicone tubing, and implanted subcutaneously (s.c.) in the interscapular space. Rats received peptide 234 (2 nmol in 6 μl/d; n = 11) or aCSF (n = 8) via the osmotic mini-pump at the rate of 0.25 μl/h for 14 days and were weighed every 3rd day. Recently, there has been a debate on the blockade of kisspeptin signaling by peptide 234 [31], nonetheless, we and others have repeatedly documented the effectiveness of this peptide as a potent kisspeptin receptor antagonist in rats [23,32,33]. Rats were monitored daily for vaginal opening and first estrous (markers of puberty onset). Correct cannula placement in the MePD was confirmed by microscopic inspection of 30 μm brain sections. Only data from animals with correct cannula placement were analyzed. Adult female rats (100 days old) obtained from Charles River were implanted with osmotic mini-pumps for bilateral intra-MePD injection of Peptide 234 (2 nmol in 6 μl/d; n = 13) or aCSF (n = 9) for a 14 day period as described above with stereotaxic coordinates being 3.14 mm posterior to bregma (AP), 3.4 mm lateral (ML) and 8.6 mm below the surface of dura (DV). Estrous cycle was monitored daily through vaginal cytology for 22 days and normal estrous cyclicity was defined as having at least 2 consecutive normal estrous cycles, which lasted for 4–5 days with 1–2 days of estrus. The cycle length was determined by the number of consecutive days from the last day of a cornified smear to the last day of an estrus smear in the subsequent cycle. Each rat was also fitted with an indwelling cardiac catheter via the jugular veins, to facilitate serial blood sampling for LH measurement. The catheters were exteriorized at the back of the head and enclosed within a 30-cm metal spring tether (Instec Laboratories, Boulder, CO, USA) secured to the slotted screw [34]. The distal end of the tether was attached to a fluid-filled swivel (Instec Laboratories), which allowed the rat to move freely around the enclosure. On the day of experimentation, rats were attached via the cardiac catheter to a computer-controlled automated blood sampling system for the intermittent withdrawal of 25 μl blood samples without disturbing the animals [35]. Blood sampling commenced at 1300 h on the day of proestrus and samples were collected every 30 min for 7 h to determine LH surges. Correct cannula placement in the MePD was confirmed by microscopic inspection of 30 μm brain sections. Only data from animals with correct cannula placement were analyzed. A double-antibody RIA supplied by the National Hormone and Peptide Program (Torrance, CA, USA) was used to determine LH concentrations in the 25-μl whole-blood samples. The sensitivity of the assay was 0.093 ng/ml. The interassay variation was 6.8% and the intraassay variations was 8.0%. Comparison between groups were made by subjecting data to one-way analysis of variance (ANOVA) and Dunnett’s test. Data are presented as the mean ± S.E.M. P < 0.05 was considered statistically significant.

Résumé de l’IA

Study Justification:

This study aimed to investigate the role of amygdala kisspeptin in pubertal timing in female rats, specifically focusing on the mechanism by which maternal obesity disrupts reproductive function in offspring. The researchers examined the expression of Kiss1, a gene involved in pubertal timing, in different brain regions of pre-pubertal and young adult rats. They also assessed the effects of kisspeptin receptor antagonist on pubertal timing, estrous cyclicity, and preovulatory LH surges in female rats.

Highlights:

1. Maternal obesity increased Kiss1 mRNA expression in the amygdala of pre-pubertal male and female offspring, suggesting a potential mechanism by which maternal obesity affects reproductive function in offspring.
2. Maternal obesity reduced Kiss1 expression in all three brain regions of 3-month-old female offspring, indicating long-term effects on reproductive function.
3. Antagonism of amygdala kisspeptin delayed puberty onset, disrupted estrous cyclicity, and reduced the incidence of LH surges, highlighting the importance of amygdala kisspeptin in pubertal timing and ovulation.

Recommendations:

1. Further research is needed to understand the specific mechanisms by which maternal obesity affects Kiss1 expression in the amygdala and other brain regions.
2. Future studies should investigate the long-term effects of maternal obesity on reproductive function in offspring and explore potential interventions to mitigate these effects.
3. Additional research is warranted to explore the role of amygdala kisspeptin in other aspects of reproductive function and its potential implications for human health.

Key Role Players:

1. Researchers and scientists specializing in reproductive biology and neuroscience.
2. Animal care technicians and facilities for breeding and housing rats.
3. Ethical committees and regulatory bodies responsible for approving and overseeing animal experiments.
4. Funding agencies and organizations that provide financial support for research.

Cost Items for Planning Recommendations:

1. Animal procurement, breeding, and housing.
2. Research equipment and supplies for molecular biology techniques, such as qRT-PCR.
3. Surgical equipment and supplies for implanting cannulae and osmotic mini-pumps.
4. Laboratory facilities and maintenance.
5. Personnel salaries and benefits for researchers, technicians, and support staff.
6. Ethical approval and licensing fees.
7. Publication and dissemination of research findings.
Please note that the cost items provided are general categories and may vary depending on the specific research institution and location.

Force de la preuve

The strength of evidence for this abstract is 7 out of 10.
The evidence in the abstract is fairly strong, but there are some areas for improvement. The study design includes both in vivo and in vitro experiments, which adds to the strength of the evidence. The use of Sprague-Dawley rats, a commonly used animal model, also adds credibility to the findings. However, there are a few areas that could be improved. Firstly, the sample size for some of the experiments is relatively small, which may limit the generalizability of the results. Additionally, the abstract does not mention any statistical analyses that were performed, which is important for determining the significance of the findings. To improve the evidence, increasing the sample size and including statistical analyses would be beneficial.

Abstrait

All procedures were conducted in accordance with the United Kingdom Home Office Animals (Scientific Procedures) Act 1986. The protocols were approved by the Committee on the Ethics of Animal Experimentation of King’s College London. Adult female Sprague-Dawley rats obtained from Charles River (Margate, UK) were used as breeders in our facility at King’s College London; producing animals for study 1 and 2. A separate group of adult female Sprague-Dawley rats (Charles River) fed a normal diet throughout their life course was used in study 3. The rats were housed under controlled conditions (12 h light, 12 h dark cycle, lights on at 0700 h; temperature 22°C ± 2°C) with ad libitum access to food and water. Female Sprague-Dawley rats were fed either a standard chow diet (RM3, Special Dietary Services, Essex, UK) or a highly palatable energy-dense obesogenic diet consisting of 20% animal lard, 10% simple sugars, 28% polysaccharide, and 23% protein (w/w); energy 4.5 kcal/g (Special Dietary Services) and supplemented ad libitum with sweetened condensed milk [∼55% simple sugars and 8% fat, 8% protein (w/w); Nestlé] and fortified with mineral and vitamin mix (AIN 93G; Special Diets Services). The animals were maintained on these diets for 6 weeks before mating, during pregnancy and lactation. The effects of these diets on maternal phenotype has been described previously [25]. Litter size was standardised to 8 pups (4 male, 4 female) 48 hours after birth. All offspring were weaned at postnatal day (pnd) 21 and subsequently fed RM1 diet ad libitum. In subsequent experimental groups, 2 males and 2 females from each litter were used. Male and female offspring of control (OffCon) and obese (OffOb) dams were culled on pnd 30 (OffCon, n = 8 per sex; OffOb, n = 10 per sex) or 100 (OffCon, n = 8 per sex; OffOb, n = 10 per sex) for determination of Kiss1 mRNA expression in ARC, AVPV and MePD. These offspring were prepubertal and without significant difference in body weight at pnd 30 as described previously [25]. Animals were killed by decapitation and brains rapidly removed, snap frozen on dry ice and stored at -80°C until processing. Brains were cut into 300μm thick coronal sections using a cryostat (Bright Ltd., Cambridgeshire, UK) and mounted on coated polysine slides (ThermoFisher Scientific, Braunschweig, Germany). Brain punches were taken using the micropunch method [26,27] with coordinates obtained from the rat brain atlas of Paxinos and Watson [28]. For both ARC and AVPV nuclei, a single midline punch (1 mm diameter) was taken from bregma -1.7 to 3.9 and 0.26 to -1.3 respectively, while bilateral punches (0.6 mm diameter) from the MePD were taken from bregma -2.2 to -3.6. The punched sections were fixed with formalin and stained with crystal violet to confirm correct punch positioning under a microscope. Total RNA was extracted from the punched ARC, AVPV and MePD tissues for each rat using TRI reagent (Sigma-Aldrich, Poole, UK) and reverse transcribed using the reverse transcriptase Superscript II (Invitrogen, Carlsbad, CA, USA) and random primer following the manufacturer’s instructions. Hypoxanthine phosphoribosyltransferase 1 (Hprt1) mRNA was used as a reference gene for normalization of target gene. The primers used for quantitative real-time PCR are shown in Table 1. The primer pairs selected were designed to amplify across at least one intron, ruling out the possibility of identical size bands resulting from genomic DNA amplification. Reaction conditions for Kiss1 were optimized to give the best results for the primer and for the different quantities of target in samples [29]. The sample cDNA prepared as above was used as a template for the PCR using the Applied Biosystems® ViiA™ 7 Real-Time PCR System. During PCR, the amplified cDNA products were detected after each annealing phase in real time using the QuantiTect fast SYBR Green kit (QIAGEN, Hilden, Germany). Each reaction included 2 μl sample cDNA (optimized so that sample values of the PCR product were within the standard curve), 0.8 μl each of 10 μM antisense and sense primers, 4 μl QuantiTect SYBR Green mix, and 0.4 μl water to give a total reaction volume of 8 μl. The PCR cycling conditions for Kiss1 were initial denaturation and activation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 sec and annealing at 56°C for 10 sec and 72°C for 10 sec. The Hprt1 reaction conditions were 5 min at 95°C for one cycle, then 10 sec at 95°C, 10 sec at 55°C, and 10 sec at 72°C for 40 cycles. Expression level of Kiss1 was determined by the threshold cycle (Ct) value in the exponential phase of the PCR reaction and normalized to the respective Hprt1 Ct value using the comparative Ct method [30]. Post-weanling female Sprague-Dawley rats fed a normal diet prior to pregnancy and during pregnancy and lactation were used to determine the effect of MePD kisspeptin antagonism on pubertal timing. All surgical procedures were carried out under a combination of ketamine (Vetalar, 600 mg/kg, i.p.; Pfizer, Sandwich, UK) and xylazine (Rompun, 60 mg/kg, i.p.; Bayer, Newbury, UK) anesthesia. On pnd 21, animals were anaesthetized and secured in a David Kopf stereotaxic frame and small holes drilled into the skull at a location above the MePD after a small incision of the scalp. A 28-gauge cannula (Plastics One, Roanoke, VA, USA) was fitted bilaterally towards the MePD. The stereotaxic coordinates for implantation of the cannulae previously optimized [24] were 2.5 mm posterior to bregma (AP), 3.2 mm lateral (ML), and 7.8 mm below the surface of the dura (DV) using the rat brain atlas of Paxinos and Watson [28]. An osmotic mini-pump (Model 2002; Alza Corp, Mountain View, CA, USA) prefilled with kisspeptin receptor antagonist (Peptide 234; Sigma Adrich) or artificial cerebrospinal fluid (aCSF) was attached to the cannula with silicone tubing, and implanted subcutaneously (s.c.) in the interscapular space. Rats received peptide 234 (2 nmol in 6 μl/d; n = 11) or aCSF (n = 8) via the osmotic mini-pump at the rate of 0.25 μl/h for 14 days and were weighed every 3rd day. Recently, there has been a debate on the blockade of kisspeptin signaling by peptide 234 [31], nonetheless, we and others have repeatedly documented the effectiveness of this peptide as a potent kisspeptin receptor antagonist in rats [23,32,33]. Rats were monitored daily for vaginal opening and first estrous (markers of puberty onset). Correct cannula placement in the MePD was confirmed by microscopic inspection of 30 μm brain sections. Only data from animals with correct cannula placement were analyzed. Adult female rats (100 days old) obtained from Charles River were implanted with osmotic mini-pumps for bilateral intra-MePD injection of Peptide 234 (2 nmol in 6 μl/d; n = 13) or aCSF (n = 9) for a 14 day period as described above with stereotaxic coordinates being 3.14 mm posterior to bregma (AP), 3.4 mm lateral (ML) and 8.6 mm below the surface of dura (DV). Estrous cycle was monitored daily through vaginal cytology for 22 days and normal estrous cyclicity was defined as having at least 2 consecutive normal estrous cycles, which lasted for 4–5 days with 1–2 days of estrus. The cycle length was determined by the number of consecutive days from the last day of a cornified smear to the last day of an estrus smear in the subsequent cycle. Each rat was also fitted with an indwelling cardiac catheter via the jugular veins, to facilitate serial blood sampling for LH measurement. The catheters were exteriorized at the back of the head and enclosed within a 30-cm metal spring tether (Instec Laboratories, Boulder, CO, USA) secured to the slotted screw [34]. The distal end of the tether was attached to a fluid-filled swivel (Instec Laboratories), which allowed the rat to move freely around the enclosure. On the day of experimentation, rats were attached via the cardiac catheter to a computer-controlled automated blood sampling system for the intermittent withdrawal of 25 μl blood samples without disturbing the animals [35]. Blood sampling commenced at 1300 h on the day of proestrus and samples were collected every 30 min for 7 h to determine LH surges. Correct cannula placement in the MePD was confirmed by microscopic inspection of 30 μm brain sections. Only data from animals with correct cannula placement were analyzed. A double-antibody RIA supplied by the National Hormone and Peptide Program (Torrance, CA, USA) was used to determine LH concentrations in the 25-μl whole-blood samples. The sensitivity of the assay was 0.093 ng/ml. The interassay variation was 6.8% and the intraassay variations was 8.0%. Comparison between groups were made by subjecting data to one-way analysis of variance (ANOVA) and Dunnett’s test. Data are presented as the mean ± S.E.M. P < 0.05 was considered statistically significant.

Matériaux

N/A

Innovations

Based on the provided information, it is difficult to determine specific innovations for improving access to maternal health. The description provided focuses on the role of amygdala kisspeptin in pubertal timing in female rats and the effects of maternal obesity on reproductive function in offspring. It does not directly address innovations or recommendations for improving access to maternal health.

To provide recommendations for improving access to maternal health, it would be helpful to have more information on the specific challenges or issues related to maternal health access that need to be addressed. This could include factors such as geographic barriers, lack of healthcare facilities or resources, cultural or social barriers, or specific health concerns related to maternal health.

Once more information is provided, it would be possible to suggest innovations or recommendations tailored to the specific context and challenges of improving access to maternal health.

AI Innovations Description

The provided description is about a study investigating the role of amygdala kisspeptin in pubertal timing in female rats, specifically focusing on the effects of maternal obesity on reproductive function in offspring. The study examined Kiss1 expression in different brain regions of pre-pubertal and young adult rats, and also assessed the impact of kisspeptin receptor antagonist on pubertal timing, estrous cyclicity, and preovulatory LH surges in female rats.

However, the description does not provide any specific recommendation or innovation to improve access to maternal health. If you are looking for recommendations to improve access to maternal health, here are a few suggestions:

1. Increase availability of maternal health services: Ensure that maternal health services, including prenatal care, skilled birth attendance, and postnatal care, are available and accessible to all women, especially in remote and underserved areas.

2. Strengthen healthcare infrastructure: Invest in improving healthcare infrastructure, including facilities, equipment, and trained healthcare professionals, to provide quality maternal health services.

3. Enhance community-based interventions: Implement community-based interventions, such as mobile clinics, community health workers, and outreach programs, to reach women in rural and marginalized communities who may have limited access to healthcare facilities.

4. Improve transportation and logistics: Address transportation barriers by improving road networks, transportation systems, and emergency referral systems to ensure timely access to maternal health services, particularly in emergencies.

5. Increase awareness and education: Conduct awareness campaigns and educational programs to empower women and their families with knowledge about maternal health, including the importance of antenatal care, safe delivery practices, and postnatal care.

6. Address socio-economic factors: Address socio-economic factors, such as poverty, gender inequality, and lack of education, which can hinder access to maternal health services. Implement policies and programs that aim to reduce these barriers.

7. Strengthen health financing mechanisms: Ensure that maternal health services are affordable and accessible to all women by strengthening health financing mechanisms, such as health insurance schemes and social protection programs.

These recommendations can help improve access to maternal health services and contribute to better maternal and child health outcomes.

AI Innovations Methodology

The provided text seems to be a scientific research paper discussing the role of amygdala kisspeptin in pubertal timing in female rats. It includes details about the experimental procedures, such as the diets fed to the rats, brain tissue collection, gene expression analysis, and the effects of kisspeptin antagonism on pubertal timing and estrous cyclicity.

However, it does not directly relate to the topic of improving access to maternal health. To provide recommendations for innovations to improve access to maternal health, we would need more information about the specific challenges or areas of improvement you are interested in. Once we have that information, we can provide targeted recommendations and discuss a methodology to simulate the impact of those recommendations on improving access to maternal health.

Auteur

Dima Health

Statistics:

Citations: 24

Identifiers:

DOI: 10.1371/journal.pone.0183596

Research Areas:

Environmental, Food Security, Genetics and Genomics, Health System and Policy, Maternal Access, Maternal and Child Health, Noncommunicable Diseases, Quality of Care, Sexual and Reproductive Health, Social Determinants

Study Countries:

Mali

Study Design:

Cohort Study

Study Approach:

Quantitative

Participants Gender:

Male & Female

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