Alterations of estrous cycle, 3β hydroxysteroid dehydrogenase activity and progesterone synthesis in female rats after exposure to hypobaric hypoxia

The underlying mechanism regulating hypoxia induced alteration in female steroid hormones is first time explored in this study. To understand the mechanistic approach, female Sprague- Dawley rats were exposed to acute and chronic hypobaric hypoxia (282 mm-Hg, ~7620 m, 6 hours, 3 and 7 days). Estrous cycle, body weight, plasma progesterone and estradiol levels, morphology, histology and two key steroidogenic enzymes: 3ß hydroxysteroid dehydrogenase (HSD) and 17ß HSD activity of ovary and adrenal gland were studied. A persistent diestrous phase and a significant decrease in body weight were found in chronic hypoxia groups. Histological study suggested degenerative changes in ovarian corpus luteum of 7 days chronic hypobaric hypoxia (7CHH) group and a declined percentage of adrenocortical cells in 3 days chronic hypobaric hypoxia (3CHH) and 7CHH groups. Plasma estradiol level was unaltered, but progesterone level was decreased significantly in all hypoxic groups. Ovarian 3ß HSD activity was decreased significantly with increasing days of hypoxic treatment along with a significantly low adrenal 3ß HSD activity in 7CHH. In conclusion, hypobaric hypoxia causes a state of low circulatory progesterone level in females likely due to the degenerative changes in the female ovarian and adrenal tissues together with low steroidogenic 3ß HSD enzyme activity.

reproductive hormonal profile, but the mechanism responsible for these changes is still unexplored. The two steroid hormones: E2 and P are accountable for growth, development and functioning of ovarian follicles and maintenance of female reproductive cycle. Steroidogenic 3ß hydroxysteroid dehydrogenase (HSD) enzyme mediates oxidative conversion of pregnenolone to progesterone 26 and is located in rat ovary and adrenal tissue 26,27 . The 17ß HSD is basically belongs to ovarian granulosa cells 28 and is responsible for synthesis of E2 29 . The response of these two enzyme activity to other stressors were reported 30,31 in which the consequence was alteration in respective hormonal levels in the circulation.
Since, hypobaric hypoxia alters female reproductive hormonal profile, the probable mechanism responsible for these changes need to be assessed. Therefore, the present study investigated the effect of exposure to hypobaric hypoxia for shorter and longer duration on steroidogenic enzyme activity, ovarian and adrenal morphology and the resulting synthesized steroid hormones in female rats. Animals. Non-mated female Sprague-Dawley rats (age: 10-12 weeks, body weight: 200-230 gm) were housed at normoxic condition and hygienically maintained under 12 h light-12h dark condition (temperature 24 ± 2 °C, humidity 55% ± 2%) in the Institute's animal house. Food and water were given ad libitum. treatment groups. The rats of experimental groups were exposed to normoxia and hypoxia following an established protocol 33 and devided into four groups. (a) Control group (C): rats were kept in Institute's animal house and were not exposed to hypobaric hypoxia (normoxic control) (25 ± 1 °C, 55% ± 2%, 12 h diurnal cycle); (b) Acute hypobaric hypoxia (AHH) group: rats were exposed to a simulated altitude of 7620 m (282 mm-Hg) for 6 hours; (c) 3 days chronic hypobaric hypoxia (3CHH) group: rats were first acclimatized to a simulated altitude of 4570 m (428 mm-Hg) for 24 hours and then were exposed to 7620 m (282 mm-Hg) for 3 days; (d) 7 days chronic hypobaric hypoxia (7CHH) group: rats were first acclimatized as before and then were exposed to 7620 m (282 mm-Hg) for 7 days. A specially designed animal decompression chamber with controlled partial pressure of oxygen was used for hypobaric hypoxia exposure. The temperature, humidity, light-dark cycle and air flow inside the chamber was maintained as 25 ± 1 °C, 55% ± 2%, 12 h light-12 h dark and 0.5 L/ min/ animal respectively, similar to the conditions of rats kept in normoxia control group. The chamber was opened for 15-20 minutes each day to change the bedding, replenish the diet, measure the body weight of the rats and to take the vaginal smear for identification of estrous cycle phases. estrous cycle recording. The vaginal smear of each rat was taken with 0.9% sodium chloride (NaCl) solution, placed on a glass slide and allowed to dry. The dried smear was then fixed with 70% ethanol and stained with 0.5% methylene blue (M6900 Sigma Aldrich) solution, following an established protocol 34 and was observed under a light microscope at a magnification of 40X to identify the phases: proestrous, estrous, metestrous and diestrous phase 34 . The rats with proper progressive cycle (a total duration of 4-5 days) were considered having regular estrous cycle and further used for hypoxia exposure.

Materials and
Blood and tissue collection. The control group as well as hypoxic group rats were weighed and anesthetized with ketamine/ xylazine (70 and 6 mg/kg, i.p., respectively) immediately after exposure. Blood was collected and the rats were perfused with ice-cold phosphate buffered saline (PBS). The ovary and adrenal glands were excised, trimmed of adherent fat, washed in PBS and weighed. One ovary and adrenal gland were fixed in buffered formaline solution for histological study and another one was snap-freezed and kept at −80 °C for enzyme activity study. plasma separation. The collected blood was centrifuged (DLAB, DM0412, United States) at 1000 × g for 15 minutes. Plasma was separated and stored at −80 °C for hormonal assay.
Gonado-somatic and Adreno-somatic index. The weight of the ovary and adrenal were used to calculate gonado-somatic index (GSI) and adreno-somatic index (ASI) by using the following formulae 35 enzyme assay. Enzyme activity of 3ß HSD and 17ß HSD was measured using a pre-established method 30 .
Statistical analysis. Data were expressed as mean ± SD. The exposed groups were compared to control group individually by using t-test (V, 6.01; Graph Pad Prism, San Diego, CA, United States) for body weight, gonado-somatic and adreno-somatic index, plasma estradiol level, plasma progesterone level, ovary and adrenal 17ß HSD and 3ß HSD activity. All experiments were performed twice on 6 animals in each group. Level of significance was tested at *p < 0.05, **p < 0.01 and ***p < 0.001 level and ns p > 0.05.

Results
estrous cycle. Each phase of estrous cycle was identified by presence of three types of cells 34 : numerous nucleated epithelial cells in proestrous phase; non-nucleated cornified cells in estrous phase; nucleated epithelial cells, non-nucleated cornified cells and leukocytes in metestrous phase and plenty of leukocytes in diestrous phase (Fig. 1). The vaginal cytology of control (C) group animals displayed regular estrous cycle characterized by progressive change of phases and having a total span of 4-5 days. The rats of 3CHH group showed normal cyclicity during their exposure period (Table 1a). However, the estrous cyclicity of 7CHH exposed rats exhibited www.nature.com/scientificreports www.nature.com/scientificreports/ a halt in diestrous phase, started from 4 th ± 1 day of the 7 days hypoxia exposure and on the day of sacrifice (i.e., the 7 th day of hypoxic exposure), most of the rats were found in the diestrous phase (Table 1b). The AHH group animals had no such changes. Table 2 depicted the changes in body weight in chronic hypoxia exposed groups. CHH resulted in a substantial significant decrease (p < 0.05) in body weight on the 3 rd day of exposure though food and water were provided plenty, and then recovered on the 7 th day (but still significantly lower than the control body weight) when compared to body weight on day 0 i.e, the day before the start of the exposure. Table 3 showed the result of Gonado-somatic index (GSI) and Adreno-somatic index (ASI) among groups. No significant alterations were found in ASI, but the GSI of 3CHH group was significantly low, compared to control group (p < 0.05).

Body weight and organ weight.
Histopathological alterations in the ovary and adrenal gland. The histological analysis of ovary and adrenal gland in comparison to control group were presented in Figs. 2a, b and 3a-d. The histological study of the ovary was done regarding two features of it: follicles (100X) and corpus luteum (CL) (40X). Figure 2a showed ovarian follicle (100X) having granulosa cells, thecal cell lining, ovum, antrum and cumulus oophorus. Normal antrum with intact oocyte, surrounded by granulosa cells and theca cells were present in control group (Fig. 2a  A). The arrangement of granulosa cells were found to be dispersed and the thecal cell lining became less dense in 3CHH and 7CHH groups. The cumulus oophorus started to disperse in the 3CHH and this change was more evident in the 7CHH group, along with a bigger and less compact antrum (Fig. 2a,E,G). Atretic follicles were present in both the acute and chronic hypoxic groups (Fig. 2a,D,F,H), but 7CHH possessed the highest numbers.
A compact structure of CL, having a former follicular cavity in the center, was seen in control and acute hypoxic groups (Fig. 2b,A,B). A newly formed CL, containing blood vessels along with an old CL was observed in the 3 days hypoxia exposed group (Fig. 2b,C). Figure 2b,D illustrated degenerated CL in 7CHH group, having numerous vacuoles in the luteal cells. Figure 3a revealed the structure of adrenal gland in hypoxic groups, compared to normoxic control group (40X). The control and the acute group showed intact medulla, having blood vessels and was surrounded by the adrenal cortex. Some vacuoles were found in medulla and to some extent in cortical cells of the 3CHH group. The medulla and cortex part of the 7CHH group was found to be more disintegrated.  Table 3. Changes in GSI and ASI in control and hypoxia exposed rats. Data is presented as Mean ± SD, n = 6, *p < 0.05, ns not significant. www.nature.com/scientificreports www.nature.com/scientificreports/ The cell count of individual layers of the adrenal cortex (400X) showed variations (Fig. 3b-d). The cell number of the layer surrounding the central medulla i.e., zona reticularis (ZR), was found to be decreased by 23.41%, 31.85% and 37.58% in acute, 3 days and 7 days chronic hypoxia group respectively, in comparison to control www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ (Fig. 3b). The cell number of intermediate zona fasciculata (ZF) layer ( (Fig. 3c) was found to be decreased by 5.69% and 37.31% in acute and 3CHH groups respectively compared to normoxic control group. The 7CHH group showed a decline of 30.62% in cell count compared to control. The outer zona glomerulosa (ZG) showed a regressive number of cells with increasing duration of hypoxia, i.e., 19.42% in acute group, 20.63% in the 3CHH group and 33.81% decrease in the 7CHH group in comparison to control (Fig. 3d). Fig. 4 have elucidated the plasma E2 level in animals after exposure to hypoxia. Both acute and chronic hypoxia exposed groups showed no significant change in E2 level in comparison to control group. The plasma P level was decreased significantly in AHH (p < 0.05), 3CHH (p < 0.01) and 7CHH (p < 0.01) groups compared to control group (Fig. 5).

Plasma steroid hormone levels -estradiol (E2) and progesterone (P).
Adrenal and ovarian steroidogenic enzyme activities -3ß HSD and 17ß HSD. Both the ovarian and adrenal 17ß HSD activity showed no significant change in the hypoxic groups when evaluated with the control group (Fig. 6). A significant decrease in ovarian 3ß HSD activity was found in AHH (p < 0.05), 3CHH (p < 0.01) and 7CHH (p < 0.001) groups compared to control group (Fig. 7). On the other hand no significant alterations were seen in adrenal 3ß HSD activity in AHH and 3CHH groups when compared to normoxic control group. The adrenal 3ß HSD activity was found to be diminished significantly (p < 0.01) in 7CHH group in comparison to control group. . Graphical representation of alteration in plasma estradiol (E2) level in normoxic control and hypoxia exposed rats. C: normoxic control, AHH: acute hypobaric hypoxia, 3CHH: 3 days chronic hypobaric hypoxia, 7CHH: 7 days chronic hypobaric hypoxia, n = 6, ns not significant. Figure 5. Graphical representation of alteration in plasma progesterone (P) level in normoxic control and hypoxia exposed rats. C: normoxic control, AHH: acute hypobaric hypoxia, 3CHH: 3 days chronic hypobaric hypoxia, 7CHH: 7 days chronic hypobaric hypoxia. Mean values are significantly different from control value by student's t-test (*p < 0.05, **p < 0.01), n = 6, ns not significant.

Discussion
A handful of work was done on females at HA regarding their altered hormonal profile, but those studies were on different populations, had different durations of hypoxic exposure and had different objectives thus provided less comprehensive information 6,19,20,[38][39][40] . Changes in circulatory hormonal level, however did not explain the underlying changes that occur in hormone-regulated female reproductive tissues. Therefore, this study was designed to illustrate the structural and mechanistic aspect of the female reproductive system, responsible for changes in sexual hormonal profile after hypobaric hypoxia exposure and we found some interesting changes which will be discussed here.
The normal reported length of rat estrous cycle is 4 days 34,41 and we have observed similar duration of the cycle (4-5 days) in control rats of our study. In other studies, the rodent estrous cycle was reported to exhibit changes in response to atmospheric pressure, temperature and stress 24,42 . Nelson and Srebnik found an elongated diestrous or estrous phase in HA (3800 m) female native rats 43 . Donayre has also reported a diminished frequency of estrous in rats after 1-10 days exposure to HA (4267 m) that revived during 11-20 days period, prolonged estrous phase was found after 21 days and the cycle became anestrous after 90 days, which finally recovered on descent to sea level 24 . Our observation seemed to be similar with these studies, as the cycle of 7 days hypoxia exposure group became irregular due to lengthened diestrous phase.
The result of the present study showed a significant fall in body weight till 3 rd day of chronic hypoxia exposure and then started to regain from 4 th day onwards. Similar loss of rat body weight exposed to 7620 m was also Figure 6. Graphical representation of alteration in ovarian and adrenal 17ß hydroxysteroid dehydrogenase enzyme activity in normoxic control and hypoxia exposed rats. C: normoxic control, AHH: acute hypobaric hypoxia, 3CHH: 3 days chronic hypobaric hypoxia, 7CHH: 7 days chronic hypobaric hypoxia, n = 6, ns not significant. Figure 7. Graphical representation of alteration in ovarian and adrenal 3ß hydroxysteroid dehydrogenase enzyme activity in normoxic control and hypoxia exposed rats. C: normoxic control, AHH: acute hypobaric hypoxia, 3CHH: 3 days chronic hypobaric hypoxia, 7CHH: 7 days chronic hypobaric hypoxia. Mean values are significantly different from control value by student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001), n = 6, ns not significant.

Scientific RepoRtS |
(2020) 10:3458 | https://doi.org/10.1038/s41598-020-60201-4 www.nature.com/scientificreports www.nature.com/scientificreports/ reported in an another study 44 . The GSI, a scale of change in gonadal weight in relation to body weight, was found to be decreased significantly (p < 0.05) in the 3CHH group compared to control group as a consequence of low body weight. The histological study of ovarian tissue of hypoxic groups showed distinct features in comparison to the normoxic group. 3 and 7 days hypoxia exposed ovary showed regressive lining of surrounding thecal cells along with less compact granulosa cells in matured follicle. The granulosa cells surrounding the oocyte i.e., cumulus oophorus, provides nutrients to oocyte, helps it to mature and is important for fertilization 45,46 . The cumulus-oocyte complex is also important for fertilization as it prevents hardening of the zonapelucida and thus facilitates sperm penetration 47 . In this study, we found that the cumulus oophorus started to denude in the 3CHH group, which was again found to be more degenerated in the 7CHH group and therefore might interfere with oocyte maturation and ovulation process. Atresia was reported to be a result of follicular apoptosis, regulated by gonadotrophins 48 . Our observation revealed the existence of some atretic follicles in all three hypoxic groups. The CL of 7 days hypoxia exposed group depicted marked degeneration of the luteal cells. In addition, the adrenal gland of hypoxia exposed groups also portrayed interesting changes like, the medulla of 3 and 7 days hypoxia group was found to be degenerated by the formation of vacuoles and these vacuolar degenerations were more in 7 days group. The steroidogenic cells of inner ZR and ZF displayed a marked decline in percentage count than the control. The cell count of ZG or zona multiformis as termed in animals 49 , was found to be decreased in all three hypoxic groups of our study. The percentage of decrease was proportional with the increasing time of hypoxic exposure. The cells of adrenal medulla secretes epinephrine and norepinephrine, ZG of the adrenal cortex is responsible for the production of aldosterone and ZF and ZR synthesizes steroid hormones 49,50 . The ovarian CL and adrenal cortical cells are great source of progesterone and deterioration of these structures in our study might have an impact on progesterone secretion as well.
The female reproductive functions are dependent on two vital steroid hormones, viz. estrogen and progesterone 51 . In the present study, plasma steroid hormones were measured and it was found that the circulatory E2 level was unaltered, but P level was decreased significantly in all hypoxic groups compared to normoxic group. Previous literatures showed that the level of E2 remained unaltered in women exposed to stress like cold pressure test for a shorter duration 52 . But in case of animals, when female rats exposed to a longer duration (21 days, intermittently) of restraint stress, their E2 level was declined 53 , suggesting that E2 level is only affected after prolonged stressor stimuli. HA hypoxia affects female hormonal level, as Gonzales found unaltered E2 and increased follicular stimulating hormone (FSH) level in Peruvian HA female natives (4340 m) in comparison to sea level residents 20 . Another study on the same population found a declined level of leutinizing hormone (LH) and estrogen in the follicular phase of HA natives 19 . A recent study reported decreased P level in sea level natives after 14 days trekking at 5050 m, though E2, FSH and LH hormones were found to be unchanged 6 . Some studies measured the reproductive hormonal level in females at HA for phasic categorization: increased estrogen and P was found in female lowlanders in follicular phase, during their 12 day residence at HA (4300 m) 38 ; a change (not significant) in luteal P level was determined on 3 rd and 12 th day of HA (4300 m) exposure, in sea level residents 39 ; a declined level of luteal P and elevated E2 level in both follicular and luteal phase was reported in lowlanders during their 12 day stay at 4300 m, compared to their sea level values 40 . Apart from hypoxia other stressors also seemed to have an impact on steroid hormones. For instance, dietary cyanogens consumption, excessive iodine dosing and protozoan infection in female rodents led to both E2 and P reduction 30,31,54 . In another study toxic chemical exposure was also found to mitigate E2 in female rats 55 . After prolonged surgical stress the rat adrenal P level was found to be declined as the corticosterone level was increased 56 . In the present study, the resulting decreased P level and persistent diestrous phase could be linked together, as Haim et al. has reported that the P took two peaks: one in proestrous (along with E2) and another in metestrous which is of low magnitude than the former peak 57 and an intact functional ovary is required in the late diestrous phase for entering into the proestrous phase 10 . In this study, a significant decrease (p < 0.01) in plasma P along with regressed corpus luteum and decreased adrenocortical cell number in the 7CHH group brought about the changes in estrous cycle phases.
Following the quantification of circulatory sex hormones, the key steroidogenic enzymes, involved in synthesis of these hormones were also studied. It was found that ovarian 3ß HSD activity was decreased significantly with increasing duration of hypoxic exposure compared to normoxic condition. In addition, a significant decrease in adrenal 3ß HSD activity in 7CHH group, therefore could be associated with the diminished level of circulatory P. Liu and his team found downregulation of 3ß HSD gene, in male rats subjected to hypoxia 58 . Histochemical study of male toad testicular localization of 3ß HSD enzyme showed decreased activity along with a decrease in testicular hormone synthesis after exposure to 7,315 m in comparison to control group 59 . A previous study found a decrease in serum testosterone concentration along with a decrease in protein expression of 3ß HSD in male mice exposed intermittently to normobaric hypoxia 60 . 3ß HSD activity was also reported to be reduced significantly in female rats fed with dietary cyanogens for a longer duration 31 . Furthermore, in our study, ovarian and adrenal 17ß HSD activity in all hypoxic groups showed no significant alterations and probably resulted in unchanged plasma E2 level as well. However, the decrease in P was probable a result of regressed CL, adrenocortical cell hypoplasia and declined 3ß HSD activity, led to cessation of diestrous phase, as the amount of P was not sufficient to take a sharp peak to initiate proestrous phase in the 7CHH group. However, when some of the 7 days hypoxia exposed rats were kept at normoxic condition, it was found that the cyclic progression was resumed and the rats were no longer arrested in diestrous phase after 1-2 days exposure to normoxia (the result is not showed here).

conclusion
In a nutshell, it is found that chronic hypobaric hypoxia resulted in irregular estrous cycle in rats due to prolonged diestrous phase which is a consequence of mitigated plasma P level. The CL of the ovary and cells of zona reticularis and fasciculata of the adrenal cortex that are thought to produce this steroid hormone along with a decreased activity of steroidogenic enzyme 3ß HSD is responsible for low synthesis of progesterone.Therefore, the findings gathered in our study as well as the physiological regimens considered above suggested the conclusion that