Abstract: Background and Objective: Infertility and maternal stress studies have become major public health problems in most societies and this has attracted urgent global attention. This study examined the modulatory role of restraint-induced stress and clomiphene citrate (CC) administration on the reproductive programming of Wistar rats. Materials and Methods: A total of 119 healthy Wistar rats weighing between 150-200 g were used and assigned into four major groups. Stress was induced for 1, 2, 4 and 6 hrs daily by exposure of the rats to a restraint plastic chamber for 1 and 2 weeks, respectively. At the end of each experimental protocol, the animals were euthanized by cervical dislocation and serum samples were collected for hormonal assay. The average gestational length, litter size and pup weight were examined. Data were expressed as Mean±SEM and mean differences were analyzed using One-way (ANOVA) and LSD post hoc Test with SPSS 23 at p<0.05 level of significance. Results: These findings showed that restraint stress caused a significant elevation in corticosterone level, while estrogen, progesterone, follicle-stimulating hormone and luteinizing hormone were significantly reduced when compared with control. Gestation length and litter size were also significantly reduced by stress while pup weights were not significantly affected. The CC increased litter size in unstressed rats when compared to litters of stressed rats that were significantly reduced, although, CC was able to increase the litter size of stressed rats towards normal. Conclusion: Evidence indicated that stress alters reproductive potential in female rats and also, reduces the effectiveness of CC in inducing ovulation.
INTRODUCTION
Infertility is one of the major health-therapeutic problems in different societies. Globally, infertility affects between fifty and eighty million couples at some point in their reproductive lives1. Approximately 8% of men seek medical help for fertility-associated problems2. An estimated 3-4 million Nigerian couples have fertility-associated problems3. Infertility affects about 20-30% of couples in Nigeria and around the world, irrespective of their race or ethnicity4. The malefactor of couple infertility is estimated to vary between 25-50%5. One of the most common causes of infertility is ovulation failure in women, which usually occurs due to malfunction of the hypothalamus, resulting in pituitary gland dysfunction in women5.
Stress, trauma, excessive weight gain or loss, anorexia or eating disorder and vigorous exercise are factors that affect the activity of the ovaries6. Stress occurs when the organism perceives a disruption or a threat of disruption of homeostasis7. Stress can be acute or chronic. In daily life, humans and animals are often confronted with situations that demand adaptation. There is stress if adaptation is with great difficulty or impossible8. The human physiological stress response involves a complex signalling pathway among neurons and somatic cells. While our understanding of the chemical interactions underlying the stress response has increased vastly in recent years, much remains poorly understood9. Experimental studies over the past decade have been carried out on stress and its impact on male and female fertility10.
The physiologic and behavioural responses to stressors are generally well known, though mainly for male adults11. Stressors may vary from life events (e.g., divorce, serious illness or death of a relative or friend) to daily hassles (e.g., domestic affairs, financial or relational problems and queuing). Different stressors may lead to reduced female reproductive hormones, gestational duration, birth weight, sex ratio and litter size of experimental animals12-14. Psychological stress could affect these factors directly or indirectly at various levels. Reports of the association between stress and female infertility are controversial. Some studies established an opposite association between stress and female reproductive parameters15, whereas, others do not show such an association16, hence, the need for this study to examine the impact of physical stress on the reproductive potential of female adult Wistar rats.
MATERIALS AND METHODS
Study area: The study was carried out in 2019 at the Laboratory of the Department of Human Physiology, Faculty of Basic Medical Sciences, College of Health Science, Delta State University, Abraka, Nigeria.
Materials used: Materials used in the study are, electronic weighing balance (G&G-301, England), centrifuge 80-2 (Techmel and Techmel, USA), stress induction plastic tubes, Enzyme-Linked Immunosorbent Assay (ELISA) analyzer, hand gloves, laboratory coat, syringe, sample container and dissecting set.
Animal procurement and handling: One hundred and nineteen adult Wistar rats were used in this study. The animals were procured from and housed in the Animal Centre, Faculty of Basic Medical Sciences, Delta State University, Abraka. The animals weighed between 150-200 g and were fed with growers marsh produced by Rainbow Feeds Sapele, Delta State and supplied clean drinking water ad libitum.
Ethical recommendations: Ethical approval was obtained from the Research and Bioethics Committee, Faculty of Basic Medical Sciences, Delta State University, Abraka, Nigeria with reference number REC/FBMS/DELSU/18/17.
Experimental design: The rats used in this study were grouped into four major groups of seven rats each. A pilot study was conducted in which rats from groups B and C, respectively were stressed for 1, 2, 4 and 6 hrs daily for one week (28 rats) and two weeks (28 rats). The essence is to determine the degree of stress induced in the rats for the different periods (1 and 2 weeks). After the pilot study, only rats in Group C were used as described in Table 1.
Induction of stress: The restraint stress model as shown in Fig. 1 was performed as described by the studies of researchers9,17. The rats were placed in plastic tubes (inner diameter, 5.7 cm and length, 20.3 cm) fixed with adhesive tape on the outside. Both ends of the tube remained open to allow ventilation. The tubes completely restricted the lateral movement of the rats and markedly reduced their front-to-back movement. Once inside the tubes, the plastic tubes containing the rats were placed in a horizontal orientation. The rats were retained inside the tubes for variable periods, depending on the protocol of the experiment. The animals were returned to their home cages immediately after exposure to stress. The control animals were maintained in their home cage throughout the experimental period9.
| Fig. 1: | Restraint chamber used for the stress induction |
| Table 1: | Animal grouping in this experiment | ||
| Animal grouping | Description | Sample size (n) | ||
| Group A | Served as control rats fed with rat chow and clean drinking water | 7 rats | ||
| Group B | Female rats were exposed to physical stress for 7 days (Rats were stressed for 1, 2, 4 and 6 hrs daily) | 28 rats divided into 4 subgroups of 7 rats each | ||
| Group C | Female rats exposed to physical stress for fourteen days consisting of: | 77 rats divided into 3 subgroups as follows: | ||
| • | Four subgroups of rats stressed for 1, 2, 4 and 6 hrs daily, respectively | • | 28 rats for each of the 4 subgroups for the time interval of stress | |
| • | Three subgroups of rats stressed for 2, 4 and 6 hrs daily, respectively for maternal outcome experiments | • | 28 rats for each of the 4 subgroups for the time the interval of stress | |
| • | Three subgroups with rats in one subgroup stressed before pregnancy and rats in the other two subgroups stressed during pregnancy | • | 21 rats for each of the 3 subgroups | |
| Group D | Stressed female rats treated with 0.013 mg kg–1 clomifene citrate | 7 rats | ||
Mating arrangement and confirmation: The male rats for mating with the female rats in Groups A, C and D were randomly selected from Group E rats. Each of the female rats was mated by a male rat in a separate cage. The rats remained together in the cage until evidence of mating was observed. Vagina swap was obtained in the early hours of the day during the mating period as described by Nwogueze et al.12. The vaginal swap was rolled over on a glass slide containing normal saline and viewed under the microscope. The day of the presence of sperm cells from the swap was considered the first day of gestation. Following confirmation of mating, the male rats were separated from the female rats and the female rats returned to the group they belonged. The following pregnancy outcomes were determined in each of the mated subgroups and recorded, length of gestation, litter size and pup weights, respectively.
Administration of drug: Induction of ovulation was achieved through the administration of clomifene citrate (Palam Pharma. Pvt. Ltd., India) at a dose of 0.013 mg kg–1 b.wt. The administration was through orogastric cannula adopting the methods of Nwogueze et al.12.
Biochemical examination: Hormonal assays were carried out using ELISA biochemical kits for serum estrogen, progesterone, LH, FSH and corticosterone (Pishtaz Teb, Zaman Diagnostics, Tehran, Iran) as described below.
Estimation of serum corticosterone: Total serum corticosterone was analyzed with ImmuChemTM Double Antibody Corticosterone 125I RIA Kit (MP Biomedicals LLC, New York, USA). The serum was diluted with phosphosaline gelatin buffer (pH 7.0+/-0.1) at 1:200. About 100 μL of this dilution was used for the assay. A total of 200 μL of corticosterone-125I label was added followed by 200 μL of corticosterone-3-carboxymethyloxime. The samples were incubated at room temperature for 2 hrs and then 500 μL of the precipitant solution was added. All of the above-mentioned reagents were provided in the kit. The samples were centrifuged at 2300 rpm for 15 min at room temperature (Eppendorf Centrifuge 5810 R, Eppendorf, Germany). The supernatant was removed from the tubes. The radioactivity of the precipitates was measured with a 1260 Multigamma II-gamma counter (LKB Wallac, Sollentuna, Sweden).
Estimation of estrogen and progesterone: Serum estradiol and progesterone was were estimated by Enzyme-Linked Immunosorbent Assay (ELISA). The present study adopted the procedure for the standard as well as experimental serum samples following the method described by Chukwuebuka et al.18.
Estimation of follicle stimulating and luteinizing hormone: Serum FSH and serum LH were estimated by Enzyme-Linked Immunosorbent Assay (ELISA). The present study adopted the procedure for the standard as well as experimental serum samples following the method described by Chukwuebuka et al.18.
Statistical analysis: Results were expressed as Mean±Standard Error of Mean. The statistical evaluation of data for significance was done using One-way Analysis of Variance (ANOVA) and Least Significant Difference Multiple Comparisons post hoc Tests. The statistical data were analyzed using the Statistical software, SPSS 23. A p-value of less than 0.05 (p<0.05) was considered statistically significant.
RESULTS
Figure 2 was obtained from the pilot study in which rats were stressed for varying periods (one and two weeks). Following stress application to the animals, serum corticosterone level was determined. Based on this result, subsequent experiments were based on two weeks duration. A significant duration-dependent increase in serum corticosterone levels of rats was observed after stress application for varying periods of one week and two weeks. Serum corticosterone level was higher for rats stressed for two weeks compared to rats stressed for one week (F-value 39.34). Significant (p<0.05) increase in Corticosterone level was observed in rats stressed for 4 and 6 hrs for two weeks compared to rats similarly treated for one week.
After 2 weeks of stress application for varying periods, a duration-dependent and significant (p<0.05) decrease in estrogen level was observed in these female rats when compared to control (F-value 20.882) as shown in Fig. 3. This reduction was however lowest in rats stressed for 6 hrs daily for the two weeks. Comparing the different duration of stress exposure of the rats, a significant decrease (p<0.05) in estrogen level was also observed in rats stressed for 6 hrs when compared to rats stressed for 2 and 4 hrs daily, respectively.
In Fig. 4, the effect of stress for 2 and 6 hrs on serum progesterone was not significantly different from that of control rats, a significant (p<0.05) but the transient reduction in progesterone level was recorded for rats stressed for 4 hrs daily for the two weeks when compared to control (F-value 4.56). This observed reduction in progesterone level reversed towards the control values after 2 hrs. A significant (p<0.05) decrease in progesterone level was also observed when rats stress for 4 hrs daily was compared with rats stressed for 2 and 6 hrs daily, respectively.
Figure 5 revealed that two varying periods of stress (2 and 4 hrs) had no significant effect on serum FSH levels compared with values obtained in control rats. However, with induction of stress for a further two hrs (6 hrs), serum FSH level was significantly (p<0.05) reduced in these stressed rats (F-value 4.033). Comparing the different duration of stress exposure, a significant reduction in the serum FSH level was observed for rats stressed for 6 hrs daily for the two weeks when compared to values obtained in 2 and 4 hrs, respectively.
| Fig. 2: | Changes in serum corticosterone level of rats exposed to stress for 1 and 2 weeks #p<0.05 compared with stress exposure for 4 hrs daily for one week and +p<0.05 compared with stress exposure for 6 hrs daily for one week |
| Fig. 3: | Changes in serum estrogen level of rats exposed to stress for 2 weeks *p<0.05 compared with the control group, ap<0.05 compared with stress exposure for 2 hrs and bp<0.05 compared with stress exposure for 4 hrs |
| Fig. 4: | Changes in serum progesterone level of rats exposed to stress for 2 weeks *p<0.05 compared with the control group, ap<0.05 compared with stress exposure for 2 hrs and bp<0.05 compared with stress exposure for 6 hrs |
| Fig. 5: | Changes in serum FSH level of rats exposed to stress for 2 weeks *p<0.05 compared with the control group and ap<0.05 compared with stress exposure for 4 hrs |
| Fig. 6: | Changes in serum LH level of rats exposed to stress for 2 weeks *p<0.05 compared with the control group |
| Fig. 7(a-b): | Changes in gestation length of rats exposed to restraint stress chamber, (a) Gestation length of rats exposed to stress for 2 weeks and (b) Gestation length of rats stressed before and during gestation *p<0.05 compared with the control group |
In Fig. 6, this study reported that serum LH level was significantly affected by stress. There was a significant reduction in the serum LH level of stressed rats. The duration-dependent reduction in LH level in these stressed rats was statistically significant (p<0.05) only after 4 and 6 hrs daily stress exposure, when compared with values obtained in control rats (F-value 6.38).
The effect of stress on the duration of pregnancy in experimental animals was shown in Fig. 7a. After the application of stress for 2 weeks at varying periods per day, it was observed that the gestation length of these rats was significantly reduced compared to control rats. The reduction in gestation length only shows statistical significance (p<0.05) in rats stressed for 6 hrs despite the marked reduction in gestation length observed at the 2 and 4 hrs stress periods (F-value 2.120). More so, the reduction in gestation length observed in this study shows no significance (p>0.05) among stressed rats at the different periods (2, 4 and 6 hrs). In Fig. 7b, it was observed that a significant (p<0.05) reduction occurred in the gestation length of rats stressed for 6 hrs before mating, rats stressed immediately after mating and rats stressed after 7 days of mating when compared with control (F-value 3.227). However, no significant difference in the gestation length was observed among rats stressed immediately after mating, rats stressed after 7 days of mating and rats stressed for 6 hrs before mating.
| Fig. 8(a-b): | Changes in litter size of rats exposed to restraint stress chamber, (a) Litter size of rats exposed to stress for 2 weeks and (b) Litter size of rats stressed before and during gestation *p<0.05 compared with the control group and ap<0.05 compared with stress exposure for 2 hrs, bp<0.05 compared with stress exposure for 4 hrs |
| Fig. 9(a-b): | Changes in pup weight of rats exposed to restraint stress chamber, (a) Pup weight of rats exposed to stress for 2 weeks and (b) Pup weight of rats stressed before and during gestation *p<0.05 compared with the control group, ap<0.05 compared with stress exposure for 2 hrs and #p<0.05 compared with rats stressed for 6 hrs |
The exposure to stress causes a significant (p<0.05) reduction in litter size of rats as shown in Fig. 8a. This reduction only observed statistical significance (p<0.05) in rats stressed for 4 and 6 hrs daily (F-value 16.484). This study also reports that stress has duration dependent impact on litter size. This significant (p<0.05) decrease in litter size is notable in rats stressed for 4 and 6 hrs daily when compared with rats exposed to stress for 2 hrs daily. More so, a significant (p<0.05) decrease in litter size is also observed in rats stressed for 6 hrs daily when compared with rats stressed for 4 hrs daily. In Fig. 8b, it was observed that no significant (p>0.05) difference occurred in the litter size of rats stressed immediately after mating and rats stressed after 7 days of mating when compared with control (F-value 21.600). However, exposure of rats to stress immediately before mating significantly decreased the litter size of the rats.
There was no significant (p>0.05) change that occurred in the pup weight of stressed rats when compared with the control rats (F-value 3.020) as shown in Fig. 9a.
| Fig. 10: | Changes in litter size of stressed rats treated with clomifene citrate (CC) *p<0.05 compared with the control group, ap<0.05 compared with Clomifene treated group and #p<0.05 compared with rats stressed for 6 hrs before mating |
However, a significant decrease was obtained when the comparison is made between stress periods. It was observed that rats stressed for 6 hrs daily showed a significant (p<0.05) decrease in pup weight when compared with rats stressed for 2 hrs daily, but not in rats stressed for 4 hrs. Figure 9b observed that, a significant (p<0.05) reduction in pup weight existed in rats stressed immediately after mating and rats stressed after 7 days of mating when compared with control (F-value 6.027). Significant (p<0.05) reduction in the pup weight of rats stressed immediately after mating and rats stressed after 7 days of mating was also observed when compared with rats stressed for 6 hrs before mating.
The administration of clomifene citrate (CC) significantly (p<0.05) increased the litter size of unstressed rats when compared with the control group as shown in Fig. 10. The results further showed that significantly reduced litter size of rats after 6 hrs of exposure to stress before mating (F-value 26.477). Induction of stress significantly reduced the effect of clomifene citrate on litter size. Treatment of stressed rats with CC was able to increase litter size towards normal, but not up to the litter size of the CC-treated unstressed rats.
DISCUSSION
In this study, the rats were exposed to stress for 1, 2, 4 and 6 hrs daily for one and two weeks, respectively. Results from the current study showed that stress causes a significant duration-dependent and significant increase in corticosterone levels in these female Wistar rats (Fig. 2). The elevated serum Corticosterone level was however higher in rats exposed to stress for 6 hrs daily. Stress increases glucocorticoid (Corticosterone) secretion by continuous stimulation of the hypothalamic pituitary adrenal (HPA) axis with a suppressed negative feedback action. An increase in glucocorticoid (corticosterone) secretion has been reported to be one of the most regular responses to stress and has often been used as a trustworthy marker for the strength of stressors, thus corroborating findings in this study19,20.
Results on the effects of stress on serum estrogen levels were controversial. Results from the present study showed that physical stress causes a significant duration-dependent decrease in estrogen level which was lowest in rats exposed to stress for 6 hrs daily for two weeks (Fig. 3). This reduction may be attributed to the impaired pathway in the synthesis of estrogen. Wilson et al.21 in their study using heat stress in dairy cows observed that plasma estrogen concentrations are reduced, an effect that was consistent with decreased concentrations of estrogen obtained in the present study. However, Wolfenson et al.22 experiments suggested that heat stress can cause an increase in peripheral concentrations of estradiol-17 between days 1 and 4 of the oestrous cycle, a report contrary to what was obtained in the present study. The difference in these results may be due to the use of different stressors, variable duration of exposure and level of recovery before re-induction of stress. The mechanisms by which stress alters the concentrations of circulating reproductive hormones were not known. However, one of the effects of stress may involve adrenocorticotropic hormone (ACTH) which increases cortisol secretion23, an action that has been reported to block estradiol-induced sexual behaviour24.
The report on the effects of stress on serum progesterone levels was controversial. However, this study reported that physical stress for 4 hrs daily causes a significant reduction in progesterone levels after two weeks (Fig. 4). These differences probably arise because of uncontrolled changes in several other factors that influence plasma progesterone levels. The result from the present study was consistent with that reported by Ronchi et al.25 but contrary to reports by the previous studies21,26-28. One proposed mechanism of reduced progesterone level in heat stress by previous studies suggested that heat stress affects the follicle which is ultimately carried over to the corpus luteum that produces progesterone. Several other studies have reported increased26, decreased25, serum LH or unchanged27 and blood concentrations of this hormone during heat stress in dairy cows28.
In females, the pituitary release of FSH is critical for follicular development12. A previous study on heat stress reported an increase in plasma concentrations of FSH in cattle, suggesting that elevation in FSH levels may be due to decreased plasma inhibin and follistatin production by compromised follicles29. However, a reduced FSH response in heat-stressed cows was observed after administration of a GnRH analogue30. This study observed that physical stress significantly decreases FSH levels after two weeks of exposure for 6 hrs daily (Fig. 5). The mechanism by which stress reduces FSH levels may be attributed to the fact that stress-induced rise in glucocorticoids suppresses GnRH secretion from the hypothalamus which further results in the reduction of serum FSH secretion from the anterior pituitary, while the result obtained from this study corroborates with the reports of Gilad et al.30 and it was contrary to the reports by earlier studies29,31.
The effects of stress on plasma LH concentrations were inconsistent. A study on heat stress observed that LH concentrations remain unchanged32, while others have reported increased LH concentrations and still others report decreased concentrations of this hormone30,33,34. This study observed that stress significantly decreases LH levels after two weeks of exposure for 4 and 6 hrs daily, respectively (Fig. 6). The result from the present study was consistent with the report by Gilad et al.30 and Lee33 but contrary to the findings of Howell et al.32 and Roman-Ponce et al.34 reported in their studies. In the present study, the reduced plasma LH concentration may be attributed to the fact that stress-induced rise in glucocorticoids suppresses GnRH secretion from the hypothalamus which further results in the reduction of serum LH secretion from the anterior pituitary.
The effects of stress on reproduction in mammals have been documented extensively in rodents35, but these studies were not always pertinent to other species. Despite extensive research, how stress impacts reproduction is not understood19. The result from the present study observed a significant reduction in gestation length in rats after exposing the rats to stress for 6 hrs daily for two weeks (Fig. 7). This result was consistent with a study by Wadhwa13, who observed that psychological stress in mother’s decrease the gestation period, which results in low birth weight. In another study, there is growing evidence that gestational stress can, in humans, be associated with an increased incidence of preterm birth and low birth weight36,37.
In the present study, a significant decrease in litter size is observed in rats exposed to stress for 4 and 6 hrs daily for two weeks (Fig. 8). Previous studies on animals showed that exposure of pregnant dam to stressful conditions often results in reduced litter size (embryo resorption), structural malformations, growth retardation, lower birth weight of the puppies and even a shift in the sex ratio38. Götz et al.14 also reported reduced litter size in rats exposed to chronic social stress (2 hrs/day) throughout gestation. Restraint stress for 2 hrs on each of the first 5 days of pregnancy can decrease average litter size in rats39. The above reports were consistent with the results obtained in this study.
Stress exposures during the prenatal period have implications for pregnancy outcomes as well as for morbidity and mortality. From this study, stress causes no significant (p>0.05) changes in the pup weight of rats exposed to stress for two weeks (Fig. 9). Similar to the findings in this study considerable bodies of evidence have supported the view that prenatal stress causes a reduction in birth weight40. Brunton and Russell,41 also reported that brief social stress during late pregnancy (10 min/day on days 16-20) in rats significantly reduced the birth weight of pups. Reduced birth weights of pups were also observed following overcrowding stress during the second half of pregnancy by Zielinski et al.42. Also, reduced litter weight has been reported in rats exposed to chronic social stress (2 hrs/day) throughout gestation14.
The result from this study showed that administration of 0.13 mg g–1 b.wt., of clomifene citrate significantly increased litter size of unstressed rats (Fig. 10). This study also reported that significant reduction in litter size after 6 hrs of exposure to stress before mating when, however, treatment of stressed rats with CC was able to increase litter size towards normal, but not up to the litter size of the CC treated unstressed rats. This finding was consistent with the findings of Chukwuebuka et al.43, who reported a reduction in litter size of pregnant stressed rats exposed to restraint, mirror and intruder stress model at 3 hrs/day for 3 weeks when compared with the litters of stressed rats that were improved when treated with CC. This study was also consistent with the report by Euker and Riegle39 that exposure to restraint stress for 2 hrs for the first 5 days of pregnancy can decrease the average litter size in rats. This was likely because Clomifene citrate has the potential for causing ovulation in these experimental animals, just like in humans. As a first-line ovulation induction agent, clomifene citrate (CC) has been used for over 40 years because it is readily available, inexpensive, well tolerated, safe and efficacious44-46.
The findings of this study have established that stress influences reproductive potential in female rats. The study also showed that gestation length, litter size and pup weight were all influenced by stress. The suppressive effect of stress on litter size was attributable to its interference with ovulation. Clomifene citrate administration was able to increase the litter size in rats by causing multiple ovulations, just as in humans. However, it was observed that the effectiveness of clomifene citrate was reduced after induction of stress. It is recommended that further study be conducted to ascertain the exact mechanism with which stress reduces the effectiveness of clomifene citrate in causing ovulation.
SIGNIFICANCE STATEMENT
Stress interferes with the regulatory response of maternal gestation in rats supplemented with clomifene citrate to induce ovulation in Wistar rats. Rats exposed to different intensities of physical stress alter reproductive hormone levels and the success of female reproductive capacities.