Assessment Of Stress On Serum Estradiol Levels In Female Subordinate Naked Mole Rats Following Isolation From Natal Colony

Objective: This study investigated the effects of isolation of subordinate naked mole rats from natal colonies on their reproductive success. The study aimed at establishing whether the reproductive suppression in subordinate naked mole rats is the outcome of social stress exerted by breeding female in the colony or other unknown environmental factors within the colony set-up. Blood samples from experimental and control groups were collected on 2nd, 4th, 6th , 8th, 12th and 20th weeks of experimental period for hormonal analysis using ELISA technique. The Statistical analysis done using student t-test at 5% signi�cance level. Results: Hormone analysis showed signi�cant change in both cortisol (t = 8.74, P=0.01) and estradiol (t=7.15, P=0.02) of subordinate isolated females. Results showed no correlation between stress and cyclicity. Conclusively, the observed reproductive suppression among subordinate naked mole rats in natal colonies is probably due to presence of queen or other reproductive aspects that may not be directly related to stress


Background
Naked mole rats (Heterocephalus glaber) are found in many parts of Makueni and Kitui Counties, in Kenya.Their reproduction is restricted to one queen and less than three males; the rest of the animals within the colony remain sterile.The proximate mechanisms of reproductive skew observed among the cooperative breeders have been studied.For instance, in Marmoset monkeys, a combination of behavioural and pheromonal signals from the dominant female bring about insu cient secretions of gonadotrophic hormones in other females [1].Similar studies on Meerkats showed that physical aggression by dominant breeder towards the subordinate females was responsible for reproductive suppression [2].The factors underlying reproductive inhibition among subordinate female naked mole rats in a natal colony remain obscure.Whether or not this inhibition results from social stress exerted by the breeding female, preference by breeding males to the queen over subordinate females or due to other ecological factors remain to be elucidated.The present study, therefore, interfered with the natal colony set up in order to explore the possibility that reproductive suppression on the female subordinate naked mole rats could be a form of stressor that impairs cyclicity, reproductive hormone synthesis and secretion and, therefore, reproductive success.Since the study considered isolation to be a form of stress, eusociality of the naked mole rats made them ideal animal model for the present study.The ndings of this study would provide the basis for studying variations in reproductive hormones which could be applicable to human beings when exposed to stress and how these contribute to trade-offs with reproduction.

Animal capture, sexing and Housing
Forty naked mole rats were captured by simple random sampling technique from different natal colonies through live traps method.Sexing was done before introduction of the animals into the arti cial colony.
Each animal was examined around the genital area, between the two ori ces, for the presence of a horizontal red line, a characteristic anatomical feature for females.The animals were marked with a tattoo ink for easy identi cation since they are homomorphic in appearance.Captive colony was made in arti cial burrow system consisting of small cubical Plexiglas chambers (20 cm x 20 cm x 20 cm) linked by transparent glass tubing of 3 m tunnel system in a dimly lit room maintained at approximately 26 0 C room temperature with humidity of approximately 60%.The animal house was cleaned daily and food supplied at the opening of the cubical boxes daily.Beddings for the animals were dry treated sawdust.

Experimental design
This comprised of three groups: two for experimental groups (singly isolated females (SIF) and female paired with males (FIP)) and non-isolated females (NIF) as controls.The experiment was carried out in two phases: acclimatization and isolation 2.2.1.Acclimatization Before acclimatization, 2 female subordinate naked mole rats were sacri ced.Blood samples were collected and appropriately preserved for later analysis.On the 2 nd , 4 th and 6 th weeks of acclimatization, 2 female mole rats were sacri ced at each stage and blood samples, were harvested for analysis.
Acclimatization covered the period between day 1 and week 6 of study while all animals were still in parent colony.

Isolation from parent colony
Isolation was done on week 7 of experimental period.Eighteen subordinate females were isolated and randomly assigned into two groups of 9 animals each in two separate cages for 14 weeks.One week 8, 2 subordinate females from each group were randomly selected, sacri ced and blood samples collected for analysis.The same procedure was repeated on week 12 and 20 of study.

Animal euthanasia
To ensure humane death, iso urane was administered through inhalation using soaked cotton wool for 30min in fuming chamber followed by decapitation.

Blood Sampling
Blood sample was collected using a 23-G needle directly from the apex of left ventricle after thoracotomy following anaesthesia with iso urane (Sundent Pharmaceutical Co., Ltd, Shanghai, China) in a sealed chamber.About 500µl of blood sample was collected into red-topped vacuitainers followed by centrifugation at 1000 rpm and serum decanted into clearly labelled ependorff tubes (by animal identity and date of collection) and subsequently frozen at -20 o C until hormone analysis.

Cortisol assay
Serum cortisol was assayed using ELISA technique according to the manufacturer's protocol.The technique applies the principle of competition for limited binding sites on the speci c antibody by both the hormone in sample and enzyme-conjugated hormone in the coated plates.First, the serum samples were thawed and homogenized by centrifugation.Twenty ve microliters of the standards, sample and quality controls were rst pippeted into the microtitre plate wells.Then 200 µl of Enzyme Antigen Conjugate was added and mixture incubated for 60 min at 25 o C followed by aspiration of contents and addition of 400 µl of Wash solution.The washing procedure was repeated twice with a soak time of 30 sec before aspiration of the wash solution.Then 100 µl of the substrate solution was added and the mixture incubated for 15 min at 25 o C. Then 100µl of the Stop solution was added followed by determination of absorbance at wavelength of 450nm within 30 min.The assay reagents (quality controls, standards, blanks and samples) were done in duplicate.The optical density of specimen was measured using HumaReader HS equipped with 450nm lters (Gessellschaft für Biochem und Diagnostica, mbH, Germany).The assay sensitivity for cortisol was 1.1-1.5ng/ml.The intra -assay coe cient of variation was less than 15% while the inter assay coe cient of variation was less than 10%.

Estradiol assay
Assay was also done by ELISA as per the protocol.After thawing reagents and specimen, twenty ve microliters of the calibrators, specimen and controls were rst pipetted into the micro plate wells.Then 100µl of the Enzyme Antigen Conjugate was added and vortex mixed.The mixture was incubated for 60 min at 25 o C. Then all the contents were aspirated followed by addition of 400 µl of Wash solution.
Washing procedure was repeated twice with a soak time of 30 sec before aspiration of the wash solution.
Then 100 µl of the substrate solution was added and the mixture incubated for 15 min at 25 o C. Then 50µl of Stop solution was added and mixed carefully followed by measurement of absorbance at 450nm within 10 min.The assay procedure was also done in duplicate.The optical density of specimen was measured using HumaReader HS equipped with 450nm lters.The assay sensitivity for estradiol was 13pg/ml.The intra -assay coe cient of variation was less than 15% while the inter assay coe cient of variation was less than 10%.

Statistical analysis
The quantitative data on hormonal levels were analysed with IBM SPSS statistics version 20 yielding both descriptive and inferential statistical data.Data on hormonal levels are presented as mean ± S.E.M.
For parametric data the statistical differences between groups were determined by paired t-test.All the tests were two-tailed t-tests and the signi cance level was set at P<0.05.

Quality Control
To ensure validity on the data for hormone assay, the controls were assayed with a calibration curve and the quality control samples were run in triplicate alongside samples and standards and the mean values obtained were within the acceptable ranges.The pipetting was done quickly and with precision of the aliquots to avoid assay drift that could in uence reliability of the results.The intra assay coe cients of variation were 3.10% for cortisol and 11.89% for estradiol.

of colony re-organization on serum cortisol
The mean serum cortisol levels during acclimation decreased signi cantly from 117.7 to 35.5ng/ml from day 1 to week 6 (t=3.49,P=0.04).After acclimation, there was 5.5% increase in the serum cortisol between week 8 and 1week 12 in SIF.The increase in mean cortisol levels in SIF was greatest at the time of isolation from 35.5ng/ml to 123.4ng/ml compared to levels between week 8 and week 12, whereby the hormone increased from 123.4 to 130.2ng/ml.However, there was 7.1% percentage decrease in serum cortisol in NIF.Similarly, in FIP, there was 9.9% decrease in serum cortisol levels between week 8 and 12.

Effects of colony on serum in
The serum estradiol levels increased signi cantly during acclimation with a mean of 14.5 ± 0.96 pg/ml (t=15.15,P = 0.001).The SIF had 6.3% decrease in serum estradiol level between week 8 and 12 compared to NIF with 14.4% increase while FIP had 1.9% increase in serum estradiol levels.By week 20, the SIF had a signi cant increase in Estradiol levels with mean values of 18 ± 2.52 pg/ml (t=7.15,P=0.02) compared to NIF with mean of 15 ± 0.58pg/ml.The mean serum estradiol levels in FIP also increased signi cantly from 16.1 to 25.0pg/ml (t=6.33,P=0.02).There was no signi cant difference in mean estradiol levels between the SIF and FIP (paired t =-1.73,P= 0.23).(Fig 2 ).

Discussion
Following isolation, the plasma cortisol levels in all the groups were generally higher compared to levels during acclimatization period implying that interference with social organisation by capture and isolation induced stress in the naked mole rats.Similar studies showed that repeated acute stress caused an increase in cortisol, as well as reproductive hormones in Wistar rats [3].The lower mean serum estradiol levels in female subordinate naked mole rats between day 1 to week 6 compared to the levels measured during isolation period agrees with the previous studies [4] where female naked mole rats exhibited increased estradiol and progesterone levels immediately after one month of removal from their colony.There was accompanying increased steroid hormone receptors in the female breeders compared to nonbreeding female subordinate naked mole rats.This was indicative of possible reproductive suppression that led to delayed puberty in female subordinate naked mole rats.In another similar study [5], low urinary oestrogen in the non-breeding females was reported, con rming lack of ovarian cyclicity and ovulation.
The lack of ovulation in non-breeding females was ascribed to inadequate LH concentration reported earlier [6].Results of the present study showed more signi cant changes in estradiol levels in both ISF and IPF by week 20, indicating a possible transformation in reproductive status of the female subordinate females.In the present study there was no established relationship between the cortisol and the Estradiol levels.This nding is at variance with a closely related research nding done on the Sprague dowry rats [7] where it was reported that increased serum cortisol levels during exercise led to a decrease in serum progesterone which indicated possible suppression of reproduction by increased cortisol levels-a clear indicator of physiological stress.This nding is also similar to that reported in another study where levels of oestrogen and progesterone were shown to decrease as the responsiveness to stress increased [8].In a previous study (Karsch et al., 2002), it was reported that interference of pulsatile release of LH is mediated by pathways that include the synthesis of prostaglandins and cortisol.The cortisol-mediated pathway reinforced the disruption, involved mostly in suppression of cyclicity during stressful events that cause activation of the HPA axis.
In conclusion, reproductive suppression of subordinate female can be ascribed to the presence of the queen or other reproductive aspects that may not be directly related to stress.

Limitations
Other reproductive hormones (progesterone and luteinizing hormones) that could possibly vary as a result of isolation were not considered.The present study did not consider vaginal smear instead it only focussed on oestrogen levels as a marker of cyclicity.

Declarations Ethical Approval
The research was approved by Biosafety, Animal use and Ethics committee, (REF: FAV BAUEC/2017/128) University of Nairobi and the animals were handled as directed by the committee using appropriate personal protective equipment (PPE).(Additional le 1)

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