The endoplasmic reticulum (ER) is the primary cell organ accountable for various particular cellular processes such as the synthesis and folding of secretory or membrane proteins, fatty acid and steroid biosynthesis, and Ca²⁺ storage (1, 2). Endoplasmic reticulum stress (ERS) is induced when there is an imbalance between the volume in the ER and the protein folding load, it causes a buildup of unfolded or improperly folded proteins (3). Numerous physiological and pathological processes, including excessive secretion requirement, damaged calcium homeostasis, changed lipid homeostasis, oxidative stress, and production of the mutant protein linked to disease all contribute to the induction of ERS (4–7). When ERS occurs, the three embranchments of the unfolded protein response (UPR) signaling are decoupled from glucose-regulated protein 78 (GRP78) and activated, which are protein kinase RNA (PKR)-like ER kinase (PERK), inositolrequiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) (8, 9). PERK phosphorylates eukaryotic initiation factor 2α (eIF-2α), which inhibits the entry of new proteins into the endoplasmic reticulum and attenuates translational initiation once activated. Phosphorylated eIF-2α activates activating transcription factor 4 (ATF4) transcriptional translations, which enhances antioxidant response and improves the folding capacity of the ER (10). Protein synthesis is inhibited by the phosphorylation of IRE1, and splicing X-box-binding protein 1 (Xbp1s) transcription factors are activated for chaperone activities (11). In theory, UPR restores ER homeostasis firstly as soon as possible by promoting protein degradation and transport, modification of misfolded proteins, and inhibition of protein synthesis, allowing the cell to resume normal physiological activities (12). However, an excessive ERS induces activation of CCAAT-enhancer binding protein homolog (CHOP), Jun N-terminal kinase (JNK), and cleaved caspase 3, resulting in cell death (13). Recent studies have revealed that the ERS response has a crucial influence on the regulation of female mammalian reproductive processes such as follicular development, follicular atresia, embryo attachment, and steroidogenesis (14, 15).

The ovaries provide two functions in the reproductive system of animals. One of them is the reproductive function, which is in charge of maturing and releasing oocytes for fertilization, and the other is the endocrine function, which is in charge of producing and secreting sex hormones including progesterone and estrogen (16). The steroid hormone synthesis and secretion is a complex process that is closely regulated and controlled by follicle-stimulating hormone (FSH), luteinizing hormone (LH), cytokines, and some protein enzymes (17). To begin, steroidogenic acute regulatory (StAR) proteins must be used to transport cholesterol to the inner mitochondrial membrane from the outer mitochondrial membrane (18). Then, pregnenolone is produced under the action of the cytochrome P450 side-chain cleavage enzyme (P450scc) from cholesterol, which starts that steroidogenesis process. The 3β-hydroxysteroid dehydrogenase enzyme (3β-HSD) converts pregnenolone to progesterone when it exits the mitochondria and enters the ER. Progesterone is released from granulose cells and transferred into thecal cells; there it is converted to testosterone under the action of the cytochrome P450 17-hydroxylase/17,20-lyase (P450c17) as granulose cells cannot produce androgen. After the testosterone has been moved back to granulose cells, the aromatase cytochrome P450 (P450arom) catalyzes them to produce estrogens in ER (19). The critical enzymes in the hormone synthesis pathway include P450scc, 3β-HSD, P450c17, and P450arom, and their mutations will cause a shortage of steroid hormones. The follicular and luteal cells are rich in ER to maintain cholesterol synthesis, and the normal function of ER provides the sufficient substrate for the synthesis of estrogen and progesterone (20). Therefore, we speculate that the UPR signaling, activated by ERS conditions, may be involved in the regulation of steroid hormone synthesis.

Muskrat (Ondatra zibethicus) is a semi-aquatic, medium-sized herbivore, which is native to North America (21). The muskrat breeds seasonally, with its sexual activity occurring from March to October (22). From March, when the breeding season begins, the scent glands of male muskrats begin to develop and secrete the musky substance, whose function is mainly to transmit excitement information through scent to induce the female muskrats to estrus (23). During the breeding season, males are in constant estrus while females are in cyclic estrus (24). The estrus cycle of female muskrats is generally 15-22 days, including 1-2 days in proestrus, 2-4 days in the duration of estrus, 1-2 days in postestrus, and 13-20 days in diestrus, and females after March have mature follicles and most stop estrus by early October (25). The gestation period of muskrats is very short, only 27 to 28 days, and each litter can give birth to 6 to 9 litters (24). A female can theoretically reproduce 20 to 25 muskrats per year. This reproductive characteristic makes the muskrat a good model for studying seasonal changes in gonadal functions. Numerous studies on muskrats have shown that the levels of steroid hormones are significantly elevated during the breeding season, accompanied by elevated levels of steroidogenic enzymes (26–28). For the past few years, a growing number of studies have shown an inextricable link between UPR signal and female reproduction (29). However, the changes in ERS-mediated UPR signals during different breeding seasons in seasonally breeding mammals are unclear. Accordingly, we investigate the expression patterns of UPR signal genes and steroidogenic enzymes in muskrat ovaries and the concentrations of steroid hormones of the female muskrats in this study, to gain in-depth knowledge of the relationship of UPR signal regarding seasonal variations in the ovarian steroidogenesis of female muskrats.

The present paper may be the initial research to take the female muskrat ovaries as the objects to examine the distribution patterns and expression of UPR signal genes and steroidogenic enzymes both from the BS and the NBS. These findings uncovered that the mRNA levels of GRP78, ATF4, ATF6, XBP1s, P450scc, P450arom, P450c17, and 3β-HSD were considerably higher in ovaries from the BS in comparison with those from the NBS, while, there were no considerable differences CHOP and CASP3 between the BS and the NBS at the mRNA level. In comparison with the female muskrats from the NBS, the concentrations of LH, FSH, 17β-estradiol, and progesterone were greater in female muskrats from the BS. Furthermore, RNA-seq data from the ovaries collected across the various breeding seasons showed clearly that DEGs mapped in a variety of pathways, including those associated with ERS and ovarian steroidogenesis signaling pathway with the aid of enrichment analysis. These results indicated that UPR signaling associated with the seasonal changes in ovarian steroidogenesis is activated in the breeding season and the delicate balance in redox regulation is important for seasonal reproduction in the muskrats.

When compared to the samples from the NBS, the ovarian size of the female muskrats was larger, as well as the ovarian weight of the female muskrats was heavier than those from the NBS in this study. According to histological observations, the ovaries of female muskrats from the BS had primary, secondary, antral, and dominant follicles, while those of ovarian tissues that were from the NBS only contained preantral follicles. This revealed that preantral follicles in the ovaries of female muskrats during the NBS could not continue to develop into post-antral follicles or even mature into corpus luteum. These findings are consistent with other seasonally breeding animals. In wild female ground squirrels, when compared to the ovaries from the BS, the number of secondary follicles, antral follicles, post-antral follicles, and corpus luteum considerably decreased in the NBS (34). The form and function of the ovaries of buffaloes were affected by reproductive status and season (35). During the period of seasonal infertility, the porcine oocytes were unable to fulfill their full developmental potential (36). The present research further provided strong evidence for the view that differences in follicular development existed between the BS and NBS in the ovaries of seasonally breeding mammals.

The UPR signaling, which protects against ER stress, is essential for animal development, female reproduction, the release of steroid hormones, and cellular homeostasis (37, 38). In this research, we observed the immunoreactivity of GRP78 localized in granulosa cells of mature follicles in ovaries from the BS, but not in granulosa cells of secondary follicles from the NBS, which is in accord with the study in the ovarian granulosa cells of the mouse (39). ER stress was activated in granulosa cells of late follicular development (large secondary, antral, and preovulatory) but not in granulosa cells of primary and small secondary follicles in mice (39). In addition, it has been demonstrated in numerous studies that persistent ER homeostasis disturbances during follicular growth and development cause the ERS response, which in turn triggers the UPR pathway, which in turn activates the apoptotic cascade. Yang et al. (40) demonstrated that ERS regulates apoptosis of mouse ovarian granulosa cells by up-regulating the mRNA level of CHOP. Similar results in goat ovaries also demonstrated that the ERS-mediated apoptotic pathway is indeed involved in the apoptosis of granulosa cells (41). In the condition of polycystic ovary syndrome (PCOS), ERS can also trigger ovarian granulosa cell apoptosis (42). In this study, the mRNA levels of GRP78 in the ovaries from the BS were strikingly higher than those from the NBS, indicating higher levels of ERS in the breeding season, accompanied by the increased level of antioxidant enzymes (SOD2, CAT, and GPX1). The transcription levels of UPR signaling genes (ATF4, ATF6, and XBP1s) were also considerably higher in the ovaries from the NS in contrast to those in the NBS, suggesting that UPR signaling was activated by ERS in the muskrat ovaries. However, the transcription levels of CHOP and CASP3 in muskrat ovaries were not obviously different between the BS and the NBS, indicating that the apoptosis level was not greater in the BS than in the NBS. A similar study was confirmed in an in vitro model of bovine oocytes, where it was shown that oxidative stress and UPR responses were exhibited in surviving blastocysts (43). It was discovered that the female reproductive system was affected by the modulation of UPR signals and ERS by means of the preservation of homeostasis in the cells and the beginning of apoptosis (44). The activation of the UPR by the modest levels of ERS in granulosa cells and/or cumulus cells may aid in the maturation of human oocytes (45). These results suggest that during normal follicular maturation and ovulation, ERS and the short-term UPR signaling as an adaptive response are expressed at higher levels during the breeding season, are beneficial and necessary in the muskrat ovaries, which may contribute to the production of large amounts of steroid hormones during the breeding season.

For the past few years, a growing number of researches have focused on the ERS in inextricable association with steroid hormones (46, 47). ERS not only regulates follicular development and atresia but is also critical for steroid hormone synthesis (47). As a functional tissue secreting hormones, the ovary is rich in the endoplasmic reticulum in follicular cells and luteal cells to maintain cholesterol synthesis, and normal endoplasmic reticulum function provides sufficient substrates for the synthesis of estrogen and progesterone (20, 48). Therefore, ERS-mediated UPR signaling may have a noteworthy effect on managing estrogen and progesterone production. In this study, in comparison with the samples from the BS, the levels of 17β-estrogen and progesterone in the serum of female muskrats from the NBS were considerably lowered, which were positively correlated with a notable decrease in LH and FSH concentrations when muskrat is in the NBS. In both cows (49) and mice (38), all three UPR signaling pathways were found to be activated in the luteal phase of the estrous cycle. The UPR activation-induced GRP78, ATF4, p50ATF6, and sXBP1 at the mRNA level were mostly maintained in functional and early regression stages of the mice corpus luteal (38). In the cows, the GRP78, ATF6, and XBP1 act as ER chaperones for initiating corpus luteal development and maintaining the corpus luteum, accompanied by elevated steroidogenic enzymes (49). Moreover, RNA-seq data from muskrat ovaries also confirmed the seasonal changes in ERS and steroid production, which revealed 2580 DEGs between the ovarian tissues from muskrats in the BS and NBS. In particular, there were 38 genes identified related to ER. Through further analysis of the intersection genes, we found that the ER-related pathways were engaged in the seasonal reproduction of female muskrats, and MAPK, PI3K-Akt, and ovarian steroidogenesis signaling pathways were obviously enriched by means of the KEGG analysis. Of which, the PI3K/Akt and MAPK are the most represented in the number of genes concerned. This suggests that Insulin or IGF-1 might be involved. In this study, the rise in ERS and UPR signaling was accompanied by the increased expressions of steroidogenic enzymes. Therefore, inferred from these results is that ERS may have an influence on controlling seasonal variations in steroid hormone synthesis and secretion in the ovaries of muskrats.

Some studies have also shown that the dynamic changes of ERS-related molecules seem to have some connection with steroidogenic enzymes, suggesting that UPR may affect the expression of steroid synthase (49, 50). The ATF6 pathway, which is activated by hCG, is crucial for the production of steroidogenic enzymes, particularly 3β-HSD, in Leydig cells (50). It was demonstrated that rat granulosa cells had a very high level of GRP78 expression and that ovulation stimulation, such as LH or hCG, caused GRP78 expression in these cells along with an up-regulation of LHR (51). It was found that the knockdown of XBP1 by RNAi in mouse granulosa cells reduced the expression level of P450arom and resulted in decreased estradiol secretion (52). The synthesis of progesterone and estradiol in mouse granulosa cells was clearly boosted by ATF6 knockdown, in keeping with the up-regulation of P450scc, StAR, and P450arom mRNA levels (53). Based on these evidences and our results, we suggest that the ovaries of muskrats are regulated by gonadotropins acting on the pituitary gland, and secrete large amounts of proteins and sex hormones, which are positively correlated with the high level of ERS-induced UPR signaling during the breeding season.

In conclusion, significant changes in steroid hormones and ERS occurred in the ovaries of female muskrats during seasonal reproduction. During the breeding season, with the continuous growth in follicular size and the massive proliferation of granulosa cells in the follicle, the high secretory demand of the endoplasmic reticulum (e.g., steroid hormones and steroidogenic enzymes) becomes one of the reasons for the increase in ERS. And ERS-induced UPR signaling was remarkably elevated in the muskrat ovaries during the breeding season to maintain cellular homeostasis. The results collected provide fresh avenues for investigating the role of ERS in female reproduction function and may reveal how it regulates reproduction in different seasons (for seasonally breeding mammals) and responds to varied influences (reproductive aging, stress, etc.). We intend to investigate the precise mechanism of the probable pathway in ERS impacts in vitro on the function of the ovary in muskrats in the future.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Ethics Committee of Experimental Animals at Beijing Forestry University.

WL and QW designed the study. WL and JW performed the experiments. WL, QG, and WX collected, analyzed, and interpreted data. WL drafted the manuscript. YH, HZ, and ZY supervised the project. QW, YH, HZ, and ZY reviewed the manuscript. All authors contributed to the article and approved the submitted version.