Cold is a necessary environmental stimulus. With rising global temperatures, the probability of working at high altitudes or in cold, polar environments increases significantly . Additionally, cold exposure for losing weight has become a behavioral trend among some female groups [27, 28], which dramatically increases the exposure of females to cold environments. Therefore, studying the changes and mechanisms of female physiological functions that are associated with cold exposure is both necessary and vital.
Our results showed that body weight gains decreased and changes were observed in the levels of serum sex hormones and ovarian hormone receptors, although the ovarian organ index did not change significantly. Additionally, serum AMH levels decreased and the numbers of primary and secondary follicles increased, after two weeks of cold exposure. AMH is one of the most effective and sensitive serological indicators of ovarian function , which directly reflects the ovarian reserve function. AMH can inhibit the recruitment of primitive follicles and the early growth of follicles by activating AMH receptor Ⅱ, preventing premature follicle failure, and AMH concentrations change with age instead of undergoing menstruation-related periodic changes. Follicle counting (FC) is used to examine the different types of follicles during folliculogenesis. Currently, AMH and FC are used to evaluate the development of follicles at different stages under various stimulation conditions [22, 23, 24]. The results of this study and our previous studies  have suggested that cold exposure can affect the metabolism of rats, resulting in reductions in weight gain and damage to the ovarian structure and reserve function, which affects follicular development.
POAT is a type of WAT that clings to the ovary. In mammals, adipose tissue exists as BAT, WAT, and beige adipose tissue. BAT produces heat, whereas WAT stores energy and participates in endocrine functions. Studies have shown that to promote bodily adaptations to a cold environment under cold exposure conditions, subcutaneous WAT, such as inguinal fat (iWAT), will brown . However, no research has examined whether gonadal adipose tissue, such as POAT, browns after cold exposure, and some researchers believe that this type of organ-associated adipose tissue does not brown . In this study, our results suggested that after cold exposure, the number of adipocyte mitochondria in POAT increased significantly, and specific genes associated with the browning of WAT, such as UCP1, PGC-1α, PRDM16, and Fndc5, were significantly upregulated. Two weeks of continuous cold exposure was found to induce browning in gonadal adipose tissues, such as POAT.
The changes that occur in the POAT after browning and the effects of these changes on the peri-ovarian microenvironment, the ovarian microenvironment, and ovarian function have not been explored. We found that after POAT browning, the expression levels of adipokines, such as APDN, Lep, and AMPK, were significantly increased, similar to the effects observed during iWAT browning [4, 31]. Our results suggested that the peri-ovarian microenvironment changes after POAT browning.
Ovarian estrogen, which is regulated by endocrine factors from the central nervous system, can affect the differentiation of female ovarian cells and plays an important role in the regulation of follicular development. E2 is the steroid hormone with the highest estrogen content and the strongest biological activity. P and T are intermediate products of E2 synthesis. Studies have shown that E2 deficiency (such as in an E2 synthesis rate-limiting enzyme knockout) causes mouse follicle development to stop during the sinusoidal follicle stage, and these symptoms can be relieved by the administration of exogenous E2 . Simultaneously, other studies have shown that E2 exerts anti-apoptotic functions and plays roles in cell protection and the regulation of lipid metabolism . FST is a nonsteroidal hormone expressed in the ovary, brain, pituitary, and adrenal gland and acts as an important local regulatory factor for ovarian follicles . FST regulates FSH secretion , promotes follicle maturation , and promotes embryo development. Jorgez et al.  found that mature follicles and oocytes in FST-knockout adult animals presented delayed maturation and development, and ovarian activity was terminated in advance. The administration of exogenous FST to bovine embryos during the cleavage stage could significantly improve the early cleavage rate. Regulatory factors, such as E2, FSH, and FST, together constitute the ovarian microenvironment. Increasingly, researchers believe that in addition to the central nervous system regulation, ovarian function is also regulated by both the ovarian and peri-ovarian microenvironment. Therefore, we speculate that the ovarian and peri-ovarian microenvironments may also play important roles in the follicular dysplasia induced by cold exposure.
Previous studies have shown that APDN, Lep, AMPK, and other adipokines can exert effects through autocrine, paracrine, and endocrine mechanisms. On the one hand, adipokines affect the development of follicles, and on the other hand, they affect lipid accumulation and adipose tissue metabolism in the ovarian microenvironment. APDN plays an important role in follicular development, by regulating cytochrome P450 cholesterol side-chain lyase (Cyplla1), acute regulatory protein (STAR), 3β-hydroxysteroid synthase (3β-HSD), 17-α hydroxylase 17, 20-lyase (Cyp17), aromatase (Cyp19) and other rate-limiting enzymes in the E2 synthesis pathway, to promote E2 synthesis. In POAT-excised mice, APDN contents decreased, and the expression levels of Cyplla1, Cyp19, and other genes were suppressed, resulting in decreased E2 expression in the ovarian microenvironment. Leptin can act on the ovary to promote the sensitivity of granulosa cells to FSH . Wang et al.  have demonstrated that POAT excision can cause a decrease in the Lep level of the ovarian microenvironment, which ultimately causes the granulosa cells to become less sensitive to FSH. In addition, in Lep-knockout mice, estrogen synthesis is reduced . These results showed that changes in APDN and Lep levels could affect the expression levels of E2 and FSH. Therefore, we detected the serum levels E2, FSH, T, and P, and examined the gene expression levels of ERβ, FSHR, and the rate-limiting enzymes of the E2 synthesis pathway in rat ovaries after cold exposure. The results showed no differences in serum P and T levels, whereas serum E2 and FSH levels increased, ovarian ERβ and FSHR expression decreased, and rate-limiting enzymes in the E2 synthesis pathway, such as Cyplla1 and Cyp19a1, were upregulated. Ahima et al.  also found that Lep treatment could significantly increase the numbers of primordial follicles, primary follicles, secondary follicles, and mature follicles in obese female mice, suggesting that Lep administration can improve the physiological functions of the ovary, to a certain extent. In this study, Lep upregulation appeared to play a similar role. Although the total numbers of follicles in rats did not change significantly after cold exposure, the numbers of primary and stimulated follicles increased significantly. These results suggested that APDN and Lep may affect follicular development and the ovarian microenvironment after POAT browning.
The development and maturation of follicles require energy, provided by various substrates (glucose, proteins, and lipids). AMPK is involved in energy metabolism, and decreased AMPK expression leads to a decrease in lipid accumulation in the ovarian microenvironment and the compensatory activation of the fatty acid biosynthesis pathway in the ovary . Simultaneously, AMPK participates in the browning of adipose tissue, through the AMPK-PGC1α-Fndc5 pathway. Shan et al. found that the browning of WAT occurred in myostatin (MST)-knockout rats. The deletion of MST led to the increased expression of AMPK protein, and AMPK indirectly activated the expression of PGC-1α and Fndc5. PGC-1α and Fndc5 are related genes that promote the expression of BAT and beige adipose tissue [39, 40]. MST is a negative regulator of skeletal muscle and inhibits AMPK protein expression [41, 42], and MST is inhibited by FST . FST is closely related to the browning of adipose tissue [21, 44]. Singh et al.  found that when FST is overexpressed in transgenic mice, the quality of BAT increased and the expression levels of proteins associated with BAT and beige fat increased in WAT . Exogenous FST can promote the expression of Fndc5 in mouse cells . In this study, after the browning of POAT, the expression level of MST was downregulated in POAT, and FST levels in the POAT and ovary were upregulated. These results suggested that FST may promote the browning of WAT by inhibiting MST and activating the AMPK-PGC1α-Fndc5 pathway.
Therefore, we believe that cold exposure causes abnormal follicular development, damages ovarian function, and induces POAT browning. POAT browning relieves the adverse effects associated with cold exposure on ovarian function, to a certain extent. Although we were unable to determine the exact timing of POAT browning and ovarian microenvironment changes, we tend to believe that after cold stimulation, mutual adjustments occur in the levels of adipokines induced by POAT browning and ovarian regulatory factors. This process constitutes one of the body’s compensation adjustment mechanisms following cold exposure. In addition, WAT, such as subcutaneous adipose tissue (iWAT) and adipose organ tissue (peri-dimensional adipose tissue), can directly sense temperature and generate heat, inducing the increased expression of UCP1 and PRDM16 (by 2–3-fold) . We speculate that the effects of POAT browning on the local ovarian microenvironment, due to changes in adipokines, may represent a regulatory mechanism, independent of central regulation. However, this hypothesis requires further verification.
Our study has several limitations. First, the relationship between POAT adipokines and the local ovarian microenvironment has not been fully elucidated and requires further investigation. Second, without dynamic observations, whether POAT browning has a definite protective effect on follicular development cannot be determined conclusively. Third, due to the lack of cold exposure intensity gradient verification, the potential and limits of POAT browning to provide compensatory protection for ovarian function under cold exposure conditions have not yet been elucidated. Future research remains necessary to clarify these issues. We hope that our research can stimulate interest in this field.