On the basis of sex: male vs. female rat adenosine A1/A2A receptor affinity

Objective To ensure reproducibility in biomedical research, the biological variable sex must be reported; yet a reason for using male (instead of female) rodents is seldom given. In our search for novel adenosine receptor ligands, our research group routinely determines a test compound’s binding affinities at male Sprague-Dawley rat (r) adenosine A1 and A2A receptors via in vitro radioligand binding studies. This pilot study compared the binding affinities of four adenosine receptor ligands (frequently used as reference standards) at male and female adenosine rA1 and rA2A receptors. Results The inhibition constant (Ki) values determined using female rats correspond well to the values obtained using male rats and no markable difference could be observed in affinity and selectivity of reference standards. For example, DPCPX the selective adenosine A1 receptor antagonist: male rA1Ki: 0.5 ± 0.1 nM versus female rA1Ki: 0.5 ± 0.03 nM; male rA2AKi: 149 ± 23 nM versus female rA2AKi: 135 ± 29 nM. From the limited data at hand, we conclude that even when using female rats for in vitro studies without regard for the oestrous cycle, the obtained data did not vary much from their male counterparts. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-023-06346-7.


Introduction
Since the discovery of receptor cloning and heterologous expression, novel compounds are evaluated at human receptors (the ultimate drug target); however, early in vivo studies are performed in rodents, generally Mus musculus (mice) and Rattus norvegicus (rats) which are common laboratory species [1,2].It is said that laboratory rats were already in use by 1850 and are most and 410 in rats with 82% amino acid sequence homology [1].
It is vital to account for sex as a biological variable to ensure reproducibility in biomedical research [13][14][15]; yet a reason for using male (instead of or in addition to female) rodents are seldom given.Indeed, most behavioral studies using rodents use male rodents only, seeing as researchers fear that hormonal changes during the oestrous cycle cause greater variability [16] (as well as increased costs) [13,17].Beery (2018) found that the ratio of male to female test subjects was 5:1 in neuroscience rodent studies [18]; furthermore, male-only studies seem to be increasing [19].Seeing as only male rats are used, female rats are more often than not culled, though the justification for culling is controversial [20].Female rodents are occasionally used because of ethical or economic reasons [16].
Behavioral studies have reported that female rodents are not more variable than male rodents across diverse biological traits [21][22][23].In a meta-analysis of neuroscience studies, Becker et al. (2016) found that even when female rats are used in neuroscience experiments (without regard for their oestrous cycle), their data is not less consistent than their male counterparts [13].As stated, similar results have been obtained for gene expression in humans versus rats [24].
It must; however, be noted that brain structure and chemistry are subject to sex differences, and so are adenosine and its receptors.For example, Yang et al. (2007) found that there are sex differences in the regulation of heart rate, body temperature, and locomotor activity caused by differences in adenosine A 1 receptor expression [25].Additionally, adenosine A 1 and A 2A receptors regulate the severity of learning deficits that accompany attention-deficit hyperactivity disorder, and those deficits vary between the sexes [26].Adenosine has also been implicated in differences in cocaine addiction between males and females, with an adenosine A 2A receptor antagonist having greater effects on motivation in females [27].Both McIntosh et al. (2010) and Pierling et al. (2021) suggested that gonadal hormones, specifically oestrogen, modulate adenosine receptor gene expression, and thus, cause sex differences in adenosine receptor function [28,29].Although little is known about the effect of sex as a biological variable on adenosine signaling (since almost all research were performed on males), Borgus et al. (2019) found that the effects of sex and female oestrous cycle differences on the frequency and concentration of spontaneous adenosine release in male and female Sprague-Dawley rats are complex, and alas, not consistent from one brain region to the next [30].
Interestingly, neurological conditions such as Parkinson's disease, depression, and dementia, among others (notably, adenosine receptors are associated with the potential treatment of these diseases), affect women and men differently; therefore, it is reasonable that rodent models of these diseases include both male and female subjects [13,21].
In the drug discovery process, in vivo animal studies follow in vitro adenosine receptor affinity and selectivity determination (if a promising drug candidate is identified).Considering the latter, in vitro radioligand binding assays utilizing rat membranes expressing adenosine receptors are relevant.This pilot study aims to determine the in vitro binding affinities of four well-known adenosine receptor ligands (often used as reference standards) at rat adenosine A 1 and A 2A receptors using male and female rat whole brain (expressing A 1 ) and striatal (expressing A 2A ) membranes.To the best of our knowledge, we compare and document for the first time the variance in the in vitro binding affinity (inhibition constant (K i ) values) of the reference standards at male and female rat adenosine A 1 and A 2A receptors.Based on these results, we may provide evidence for the use of both male and female rats for in vitro testing of adenosine receptor ligands.

Materials and methods
All reagents and solvents were commercially available.

Membrane preparation
The North-West University Animal Care, Health and Safety Research Ethics Committee (NWU-AnimCare) approved the study and subsequent collection of tissue samples from adult male and female Sprague-Dawley rats for radioligand binding studies (application number NWU-00035-10-A5).The research was performed in accordance with the guidelines of the South African National Standard (SANS) document (The care and use of animals for scientific purposes).Sprague-Dawley rats were sourced from the NWU Vivarium (six-week-old, (193 ± 11.94 g).Rats were housed in medium poly-carbonated cages (2 rats per cage, male and female rats were housed separately) in a well-ventilated room at a temperature of 22 ± 2 °C and relative humidity of 50 ± 10% with a with 12 h light-dark cycle.Commercially available rat chow and tap water were provided ad libitum.Upon euthanasia by decapitation, 20 male and 20 female Sprague-Dawley rats were dissected and 10 male and 10 female whole brains (excluding brainstem and cerebellum) or 10 male and 10 female striata were collected and pooled separately based on sex and whole brain or striata.(Please note that rats were not treated prior to euthanasia.)Rat brain membranes were prepared and stored as described in literature [31].The protein content of male and female rat whole brain and striatal membranes was determined using Bradford reagent and bovine serum albumin as reference standard [32].

Results and discussion
Four adenosine receptor ligands frequently used as reference standards were investigated in vitro at rat adenosine A 1 and A 2A receptor subtypes in male or female Sprague-Dawley rats' whole brains (expressing A 1 ) or striata (expressing A 2A ) using previously reported radioligand binding assays.The four reference standards include CPA, caffeine, DPCPX, and istradefylline.Before conducting the experiments, a literature search for K i values was performed: the K i values of the reference standards have been repeatedly determined at male rat adenosine receptors; however, no study reports using female rats (if the sex is at all reported).Table 1 summarized the literature K i values (male) and newly determined values using female rat whole brain (expressing A 1 ) and striatal (expressing A 2A ) membranes, respectively.The K i values determined using female rats correspond well to the values obtained using male rats.No markable difference could be observed in the affinity and selectivity (see selectivity index Table 1) of CPA, caffeine, DPCPX and istradefylline for the adenosine A 1 and A 2A receptors.
The pK i values of reference compounds at male and female adenosine A 1 and A 2A receptors were also compared, and a correlation coefficient was calculated.The R squared of the linear regression analysis is equal to the correlation coefficient (Fig. 1).It was found that male and female showed good correlation; with R squared values above 0.99 (i.e.>99%).
Additionally, the protein content of male and female rat whole brain and striatal membranes were almost identical as determined by a Bradford protein assay.Protein content male rA 1 : 6.91 mg/mL & rA 2A : 6.93 mg/mL; female rA 1 : 6.13 mg/mL & rA 2A : 6.81 mg/mL.

Conclusion
From the limited data at hand, we conclude that even when female rats are used for in vitro (and not necessarily in vivo) studies without regard for the oestrous cycle, the obtained data is not more variable than that of their male counterparts.Indeed, the use of both male and female rats would be more ethical (by reducing the number of female Sprague-Dawley rats culled, in line with the 3 Rs: Replacement, Reduction and Refinement) as well as economical; furthermore, inclusion of both sexes in basic and preclinical research could lead to significant discoveries.

Limitations
Although previous in vivo studies and the present in vitro study reported that female rodents are not more variable than male rodents across diverse biological traits, understanding sex differences and the influence of the female oestrous cycle is important for the design of effective treatments manipulating adenosine and its receptors.It must be noted that more standard adenosine receptor ligands should be compared to corroborate our findings, seeing as this pilot study merely presented the possibility of using female rat brain membranes for in vitro studies.

Fig. 1
Fig.1Correlation of pK i values at male and female rat adenosine A 1 (A) and A 2A (B) receptors

Table 1 K
i values (nM) of reference standards at male and female rat adenosine A 1 and A 2A receptors