TITLE: Exposure to Bisphenol-A and Melatonin altered the Expression of Estrogen and Androgen Receptors in Neonatal Female rats
AUTHORS: Eniola Risikat Kadir

[DESCRIPTION]
This protocol details about grouping and breeding of animals, sample collection and processing, hormonal assay, biochemical assay and quantitative real-time polymerase chain reaction procedure.


[GUIDELINES]
The animals are treat humanely with regard to following the standard guidelines for use and care of experimental animals. The research obtained a protocol approval from theUniversity of Ilorin, Ilorin, Nigeria by the Ethical Review Committee (UERC/ASN/2018/1154). This research was conducted in the Animal Holding of the College of Health Sciences, University of Ilorin.

[STEPS]
SECTION: Grouping and breeding of animals
1. 
 

Cage adult male wistar rats of proven fertility overnight with females in proestrous phase of the cycle. 
SECTION: Grouping and breeding of animals
2. Gestational Day 1 (GD 1) is the day detect sperm cells in the vagina. Once pregnancy is confirmed, isolate the pregnant rat  in a cage to stay until parturition, with a total of 12 pregnant females being used for the study. 
SECTION: Grouping and breeding of animals
3. After parturition, recognise and group the female litters . These are the second-generation Wistar rats. They have daily subcutaneous injections from post-natal day 0 for four days (PND 0 - 3). The seven groups are administered as follows:

I – Normal saline
II – sesame oil and ethanol (vehicle)
III - melatonin only (10mg/kg)
IV – 25mg/kg BPA
V – 25mg/kg BPA + melatonin (10mg/kg)
VI –50mg/kg BPA
VII – 50mg/kg BPA + melatonin (10mg/kg).
SECTION: Grouping and breeding of animals
4. Perform all administrations in the morning, and allow all the groups to mature till adulthood day 120 ± 3 days, when they are sacrificed in the morning, between 7 a.m. and 9 a.m.
SECTION: Sample collection and processing
5. After administration of BPA and melatonin treatments, the animals are left until day 120 ± 4 days. They are euthanised by administration of 20 mg/kg body weight of ketamine intramuscularly.
SECTION: Sample collection and processing
6. Following the incision and lateral reflection of the scalp, expose the skull promptly with tissue forceps to extract the entire brain and pinpoint, collect, and place the hypothalamus, situated alongside the pituitary gland at the floor of the sella turcica) into cryovials preloaded with RNA, then they are subsequently kept frozen in liquid nitrogen tanks.
SECTION: Sample collection and processing
7. The harvested brains are the nutilized for genetic (RNA) studies. Additionally, collect the female gonads; the left ovary are for follicular count on histology, which is fixed for 48 hours in 4% paraformaldehyde, and the right ovary of each animal is homogenized in 0.25 Molarity (M) ice-cold phosphate buffer followed by centrifugation of homogenates for enzyme studies.

SECTION: Sample collection and processing
8. Perform staining of the ovarian tissues in paraffin wax embedded sections according to Canene-Adams, 2013 and Haematoxylin and Eosin staining by the protocols outlined by Fischer et al., 2008. The various follicles (primary, secondary, preantral, antral, and corpora lutea are identified, counted then recorded.
SECTION: Hormonal Assay
9. Obtain the blood samples from the apex of the heart for hormonal analysis using 5 mL needles and syringes. 

SECTION: Hormonal Assay
10. Centrifugation for 3000 rpm, 15 minis used to separate the serum. 

SECTION: Hormonal Assay
11. Assay plasma luteinizing hormone, follicle stimulating hormone, oestrogen, progesterone, testosterone, and Anti-Mullerian Hormone concentration measurements using the Enzyme-Linked Immunosorbent Assay (ELISA) technique and purchase ELISA kits from Monobind Inc. Lake Forest, USA, following manufacturer's guide.
SECTION: Biochemical assay
12. 
 

This involved measuring the levels in an acidic environment, where use nitrite, upon formation, to create nitrous acid, which react with sulfanilamide to form a product. This resulting product is then coupled with N-(1-naphthyl) ethylenediamine, to give an azo dye displaying a vibrant reddish-purple color, quantifiable at 540 nm. 
SECTION: Biochemical assay
13. 
 

A unit of GPx activity is defined as the enzyme quantity necessary to catalyze the oxidation of 1 nanomolar (nM)NADPH per minute at 25 °C.


SECTION: Biochemical assay
14. 
 

The SOD enzyme's capability to impede the phenazine methosulfate-mediated reduction of nitro-blue tetrazolium dye is measured by monitoring absorbance at 560 nm over a 5 min period at 25 °C.

SECTION: Quantitative real-time polymerase chain reaction procedure
15. Euro Gold Tri-Fast solution (Euro Clone) is used to prepare the RNA. 
SECTION: Quantitative real-time polymerase chain reaction procedure
15.1. The tissue is then pulverized using a tissue homogenizer, followed by DNase treatment on the extraction samples of total RNA, such that DNA contamination from the total RNA prepared could be eliminated. 
SECTION: Quantitative real-time polymerase chain reaction procedure
15.2. Purification (through acid phenol-chloroform), precipitation, and suspension of the RNA in distilled water (dH2O) are then carried out.

SECTION: Quantitative real-time polymerase chain reaction procedure
16. The reverse transriptase enzyme (Invitrogen) is used for reverse transcription of total RNA, using M-MLV reverse transcriptase (Invitrogen), which involves retrotranscription of 1 µg of total RNA to quantify mRNA expression in experiments. 
SECTION: Quantitative real-time polymerase chain reaction procedure
17. A gently mixing of the samples through up and down pipetting and then incubate at 37 °C. M-MLV RT is inactivated for 15 min at 70 °C. Keep cDNA  at -20 °C.

SECTION: Quantitative real-time polymerase chain reaction procedure
18. NanoDrop™ 1000 spectrophotometer is used to measure cDNA and RNA concentrations. 
SECTION: Quantitative real-time polymerase chain reaction procedure
19. Nucleic acid quality is noted via absorbance ratios at 260 nm/280 nm and 260 nm/230 nm. 
SECTION: Quantitative real-time polymerase chain reaction procedure
20. Measurement of the relative amounts of the transcript of a specific gene is performed using a qRT-PCR, via a two-step procedure using:

Sybr green supermix (Biorad)- the amplification reaction and generation of melting curves of the amplicons subsequently. 
Verification of the melting curve data is running through the PCR product on 2% agarose gel.
SECTION: Quantitative real-time polymerase chain reaction procedure
21. The efficiency of the Primer is tested in reactions with six serial 1:10 dilutions of cDNA as a template to perform a calibration curve.

  
 
  
SECTION: Data analysis
22. Collect the data and analyze by two-way analysis of variance (ANOVA) and subsequently subjecte to Tukey’s (HSD) test of multiple comparison using GraphPad Prism v.6 (GraphPad Software, Inc., La Jolla, CA, USA). 

Express data as means ± SEM (standard error of mean). 
Statistical significance is taken as P value less than 0.05 (p<0.05).