Among the animal models of endometriosis used in existing studies, the rat autologous transplantation model is the most commonly used. This is due to the high success rate of the model, accurate positioning, weak immune rejection, and easy observation. The autologous transplantation model is a peritoneal endometriosis model, in which two endometrial tissue sheets were sutured to the abdominal wall to form ectopic endometrial lesions. This model is difficult to simulate the natural adhesion process of endometrial cells, and endometrial cells will not attach to other pelvic organs such as ovaries and fallopian tubes. Therefore, the influence of this model on the ovaries is bound to be in an indirect way. Some scholars believe that peritoneal endometriosis may affect the ovarian reserve function by affecting the inflammatory microenvironment in the peritoneal fluid [5]. In view of the abnormalities of hormones, cytokines, inflammatory factors and oxidative stress markers in the blood of patients with endometriosis [19], we speculate that peritoneal endometriosis may also affect the ovaries through blood or even nerves (pituitary-gonadal axis).
Endometriosis is an estrogen-dependent, chronic inflammatory disease. Local estrogen synthesis is often increased in ectopic endometrium for the growth of endometriotic lesions depends on estrogen [20]. However, studies have shown that estrogen levels in serum and follicular fluid are significantly lower in patients with endometriosis than in normal subjects [7, 17, 18] even though endometriotic lesions contain major enzymes of the estrogen synthesis pathway that can locally complete cholesterol to estrogen synthesis [21]. Thus, changes in estrogen levels may contribute to the pathogenesis of endometriosis and endometriosis-related infertility. In female mammal ovaries, estrogen synthesis is mainly dependent on theca cells and granulosa cells. In theca cells, cholesterol is transferred from the outer mitochondrial membrane to the inner membrane via Steroidogenic Acute Regulatory protein (StAR), catalyzed by P450cholesterol Side-chain Cleavage Enzymes (P450scc, the coding gene is Cyp11a1) to form pregnenolone, and then catalyzed by 3β-Hydroxysteroid Dehydrogenase (the coding gene is Hsd3b1 or Hsd3b2) and 17α Hydroxylase (the coding gene is Cyp17a1) to form androstenedione. Androstenedione enters granulosa cells and is catalyzed by Cytochrome P450 aromatase (the coding gene is Cyp19a1) to form estrogen. Notably, changes in estrogen production usually depend on the amount of pregnenolone synthesized, therefore, the transfer of cholesterol to P450scc belongs to the rate-limiting step of estrogen synthesis. In our study, we found that mRNA levels of Cyp11a1, Cyp17a1 and Cyp19a1 were significantly lower in endometriosis rats than in controls, which would affect estrogen synthesis in endometriosis rats in the long run, these results were confirmed in human blood and follicular fluid [7, 17, 18]. Furthermore, we hypothesize that reduced estrogen synthesis in the ovary may be a way for the body to defend itself against the damage caused by estrogen-rich endometriosis lesions.
Granulosa cells are not only involved in the synthesis of estrogen, but also in oocyte growth, differentiation, follicular cavity formation and follicular development, which are essential for maintaining ovarian function. Follicle-stimulating hormone (FSH) is necessary for the maintenance of normal granulosa cell function. FSH can not only promote the growth of granulosa cells and inhibit the apoptosis of granulosa cells, but also activate the cytochrome P450 aromatase of granulosa cells to promote the synthesis and secretion of estradiol [22]. In addition, for activating follicles, whether FSH can meet the needs of granulosa cells will determine whether the follicle is atresia or develops into a dominant follicle [23]. Our study found that the expression of FSHR in the antral follicles in the ovaries of endometriosis rats was significantly lower than that of the control group, which would affect the sensitivity of granulosa cells in response to FSH signals, resulting in decreased estrogen synthesis in the ovaries of endometriosis rats. From a more far-reaching perspective, the reduction of FSHR expression will also affect the development of follicles in endometriosis rats. Although it has not been verified in our research, we can obtain these results from other studies [24, 25].
The cytoskeleton is the fiber network structure of proteins in cells, in which microtubules are involved in various biological processes such as cell morphology maintenance, material transportation, information transmission and cell division. Through the GO enrichment analysis of DEMs, we found that the cytoskeleton, especially microtubule-related genes, including Dnah2, Dnah1, Dynlrb2, Krt18, Aurkb Drc1, Kif19, Agbl2, Dnah6, Wdr63, Dnah9, Drc7, Kif27, Kif6, Kif5c, and Kif9, were significantly downregulated in endometriosis rats, suggesting that endometriosis may affect ovarian structure and function by affecting the cytoskeleton of ovarian cells. In support of our findings, it has been found that the dynein light chain roadblock-type (DYNLRB) family affects zebrafish follicle development by influencing TGFβ signaling [26, 27]. High expression of AGBL2 can lead to poor prognosis in patients with ovarian carcinoma [28]. Keratin type I cytoskeletal 18 (KRT18) inhibits the apoptosis of ovarian granular tumor cells by inhibiting Fas signaling [29]. Aurora Kinase B (AURKB) and Progesterone Receptor Membrane Component 1 (PGRMC1) are jointly involved in late events of bovine granulosa cell mitosis and oocyte meiosis [30, 31]. Although it is not clear which type of cell has an abnormal cytoskeleton, existing studies have shown that endometriosis affects the cytoskeleton of the oocyte and thus affects its meiosis [32, 33]. Abnormal microtubules can also cause changes in the morphology of granulosa cells and theca cells, thereby affecting the synthesis of steroid hormones [34-37]. Microtubules may affect the synthesis of steroid hormones by affecting the transfer of cholesterol to mitochondria [38]. The above results suggest that endometriosis may affect steroid hormone synthesis and oocyte maturation by affecting the cytoskeleton structure of ovarian cells. In addition, the significant down-regulation of dynein (including Dnah2, Dnah1, Dnah6, Dnah9, etc.), kinesin (including Kif19, Kif27, Kif6, Kif5c, Kif9, etc.) and extracellular transport-related genes, up-regulation of proteasome-related genes and down-regulation of estrogen signaling-related genes in endometriosis rat ovaries suggest that endometriosis affects the microtubule structure and microtubule-related motor protein activity of ovarian cells, which affects extracellular transport, resulting in abnormal protein aggregation and increased proteasome activity, and thus damages the structure and function of ovarian cells.
The types of cell junction mainly include tight junction, adhesive junction and communicating junction. Among them, gap junctions in communicating junction have been confirmed to play a central role in the maturation and fertilization of oocytes [39]. However, some studies have also found that tight junctions play an important role in the formation of the follicle cavity and the development of the follicle [40]. Although there are differences between species, tight junction related proteins, mainly including cingulin (CGN), claudin (CLDN), occluding (OCLN) and tight junction protein (TJP), are widely present in granulosa, theca externa and thecal vascular endothelial cells, and the expression of tight junction proteins is regulated by estrogen and androgens [41-43]. During the development of marmoset follicles, the expression level of tight junction related protein CLDN5 increases in the theca cells [43]. The Cldn11 mRNA level of large antral follicles was also found significantly higher than that of small antral follicles in cattle [44]. The expression of Ocln was more prevalent in bovine theca cells than in granulosa cells and gradually decreased with follicular development [42]. These studies suggest that tight junctions are involved in follicular development. In addition, overexpression of Cldn3 [45], Cldn4 [46] and Cldn7 [47] leads to the development of ovarian cancer and malignant metastasis, thus homeostasis in tight junctions are essential to maintain normal ovarian structure and function. Through the KEGG enrichment analysis of DEMs and the GSEA analysis of the transcriptome expression profile, we found that the expression of tight junction-related genes, including Cldn3, Cldn4, Cldn7, Cldn8, Cldn10, Cldn22, Ocln, Tjp3, was significantly down-regulated in the ovaries of endometriosis rats, as compared to the control group. So, we speculate that the decreased estrogen synthesis in the ovary of endometriosis rats will affect the tight junctions between ovarian cells, resulting in abnormal follicular development and damage to ovarian function. In addition, tight junctions are also involved in the formation of the blood-follicle barrier (BFB). Structural abnormalities of BFB caused by down-regulation of tight junction related proteins expression may lead to a large number of factors in the blood to enter the follicular cavity and damage oocytes or granulosa cells.
The ectopic endometrium forms lesions by adhering to specific tissues. Through RNAseq, we did not find that the ovarian samples of rats with peritoneal endometriosis had enriched cell adhesion-related pathways, and there was no discovery to support that the endometrial cells had invaded in the ovaries in our model. In this way, we believe that endometriosis lesions can affect ovarian steroid hormone secretion through indirect pathways, such as neuroendocrine regulation, which gives an explanation for mild infertility owing to peritoneal endometriosis.