Functional role of autophagy in testicular and ovarian steroidogenesis.

Across various animal species, from Drosophila to mammals, autophagy is highly conserved being a critical player in the physiology of ovaries. In female physiology, autophagy emerges as a critical factor governing ovarian function. Specifically, critical for the survival of germ cells in development is autophagy, occurring before the development of the primordial follicle pool ( Gawriluk et al., 2011 ). Initially, follicle atresia was attributed solely to apoptosis, however, recent discoveries suggest a more nuanced mechanism. For example, evidence indicates that human granulosa cell death is orchestrated through lectin-type ox-low density lipid (LDL) receptor (LOX1)-dependent oxidized LDL (oxLDL)-induced autophagy, suggesting the involvement of autophagic pathways in programmed cell death as shown in Figure 4A ( Duerrschmidt et al., 2006 ; Serke et al., 2009 ). This finding potentially elucidates the heightened infertility rates observed in obese women, who exhibit elevated oxLDL levels ( Mutlu-Türkoğlu et al., 2003 ) and an increased prevalence of autophagic granulosa cell death ( Duerrschmidt et al., 2006 ). Mechanistic insights into autophagy-mediated steroidogenesis in ovaries. (A) Lectin-type oxLDL receptor (LOX1)-dependent oxidized LDL (oxLDL) in granulosa cell death through autophagy emphasizes its connection to obesity-related infertility. Additionally, it explores the age-associated decline in reparative autophagy, aligning with increased ROS levels and reduced LC3-II in older women. (B) Mitophagy-related proteins during luteal deterioration and highlights the impact of LDL on luteal development in vitro . (C) Beclin-1 expression and autophagy in granulosa cell longevity, especially in conditions of prolonged corpus luteum existence. (D) HD-sEVs from bovine follicular fluid on autophagy and mitophagy in bovine granulosa cells emphasize their role in reducing apoptosis and enhancing estradiol secretion through PI3K/Akt/mTOR signaling pathways. Furthermore, elevated levels of ROS, resulting from LOX1 activation by oxLDL, can induce oxidative stress-induced apoptosis. Conversely, younger women with a normal weight seem to activate reparative autophagy as a response to reduced concentrations of ROS, thereby alleviating apoptosis, thus, supporting cell survival ( Vilser et al., 2010 ). However, research by Tatone et al. (2006) and Vilser et al. (2010) suggests an age-associated decline in reparative autophagy, culminating in granulosa cell apoptosis. This decline aligns with observed increases in ROS levels and reductions in LC3-II in follicular cells from older women Figure 4A . Together, these discoveries align with the extensively recorded decrease in female fertility linked to the aging process ( Carbone et al., 2003 ; Tatone et al., 2006 ; Tatone et al., 2008 ). In animal models, these follicular cells have shown to undergo autophagy-mediated apoptosis in response to several stimuli, including nutritional scarcity and, more recently, exposure to cigarette smoke. These results suggest a plausible clarification for the established link between female infertility and smoking ( Gannon et al., 2013 ). Following ovulation, the ovary produces the corpus luteum (CL). If conception does not take place, the CL regresses by the conclusion of menstruation. Electron microscopy studies have shown the accumulation of autophagy-related bodies, e.g., autophagosomes, in various cells during CL degeneration ( Del Canto et al., 2007 ). At a molecular level, the human ovary possesses a specific voltage-gated Na channel that, when activated, starts downstream signaling leading to autophagy during CL regression ( Bulling et al., 2000 ). Conversely, higher levels of Beclin-1 expression have been observed in pregnant CL cells and under pathological conditions where the CL persists longer than usual, suggesting a role for autophagy in granulosa cell longevity ( Gaytán et al., 2008 ). Thus, we can conclude that autophagy has a dual role in regulating CL dynamics, as shown by a comprehensive review of both human and animal studies ( Gao et al., 2020 ), highlighting its significance in ovarian function. Recent studies indicate that the Akt/mTOR signaling pathway regulates the activation of ATGs in the CL. Furthermore, autophagy appears to influence progesterone synthesis by modulating the lipid droplet pool in luteal cells during pregnancy in rats. Notably, as luteal deterioration commences, there is an upregulation of mitophagy-related proteins, potentially essential for maintaining mitochondrial homeostasis ( Figure 4C ). These insights contribute to a more comprehensive grasp of the function of autophagy in the luteal changes of mammalian ovary in vivo in Sprague-Dawley rats ( Tang et al., 2019 ). Moreover, in cultured bGCs, previous trials have shown that LDL upregulates StAR mRNA and protein ( Figure 4B ). Additionally, it promotes the production of progesterone and cholesterol side chain cleavage cytochrome P-450 (CYP11A1) mRNA. Furthermore, LDL considerably increases the lysosome count in GCs. However, the inhibitors of lysosomes, such as CQ effectively mitigate these effects induced by LDLs. These findings signify that in bGCs, LDL stimulates StAR expression, progesterone production, and lysosome development while lysosomes facilitate this process via secreting FC molecules from the breakdown of LDL ( Zhang et al., 2015 ). Recently, density gradient ultracentrifugation successfully isolated HD-sEVs from bovine follicular fluid (BFF) ( Wang et al., 2023 ). These HD-sEVs induce autophagy in bovine bGCs by upregulating Beclin1 mRNA and protein expression, as well as increasing the LC3II/LC3I ratio ( Figures 4B, D ). Conversely, they suppress p62 mRNA and protein expression. HD-sEVs elevate the protein and mRNA levels of VDAC1, CTSD, and HSP60, subsequently promoting mitophagy in bGCs (as shown in Figure 4D ). Flow cytometry results indicate that HD-sEVs diminish bGC apoptosis rates by upregulating steroidogenic proteins and mRNAs, including CYP19A and HSD3B in bGCs, HD-sEVs stimulate estradiol secretion. Additionally, HD-sEVs decrease the p-mTOR/mTOR ratio and boost autophagosome production and mitochondrial structural alterations in bGCs ( Wang et al., 2023 ). The introduction of wortmannin reverses these observed effects. Mutually, BFF HD-sEVs enhance macroautophagy and mitophagy in bGCs, inhibit apoptosis in bGCs, and elevate 17β-estradiol release via the PI3K/Akt/mTOR signaling pathways as shown in Figure 4D ( Wang et al., 2023 ). Pups of Mus musculus with ATG5 -knockout ovaries showed normal follicular development but lacked CL, displaying elevated atretic follicles, showing the unsuccessful release of an egg. Additionally, these pups showed a compromised ability of the uterus to produce the endometrial gland ( Yoshii et al., 2016 ). These observations imply that autophagy is a vital player in the proper sexual development. To generate mice with ovarian-specific conditional knockout (cKO), Beclin1 -knockout mice were employed, where Beclin1 was selectively deleted in granulosa and luteal cells ( Gawriluk et al., 2014 ). This mouse model showed a nearly 75% reduction in Becn1 levels, with p62 accumulation observed in GCs. Although ovulation, fertilization, and implantation appeared similar to controls concerning reproductive phenotypes, the targeted elimination of Beclin1 led to a rise in miscarriages or premature births attributable to the failure of mitochondria to produce progesterone. Pathologically, ovaries with Becn1 -knockout lacked neutral LDs responsible for progesterone production in luteal cells. Notably, these luteal cells exhibited many large autophagosomes, suggesting compromised autophagy processes, despite Beclin1’s role in nucleation—the initial phase of autophagy. The exact mechanism through which Becn1 downregulation increases autophagosome quantities in luteal cells remains unclear. In contrast, the overexpression of BECN1 in cultured GCs stimulated progesterone synthesis by boosting the production of synthesizing enzymes, including CYP11A1, 3β-hydroxysteroid dehydrogenase, and StAR protein ( Ding et al., 2021 ). Recent research also suggests that FSH, regardless of the traditional steroidogenic pathway, enhances autophagy by upregulating Beclin1 through the PI3K/JNK/c-Jun pathway, promoting LDs breakdown in pig GCs ( Liu et al., 2021 ). Furthermore, emerging evidence highlights the significance of autophagy in melatonin-mediated regulation of progesterone release in the sheep CL ( Duan et al., 2024 ). FSH from pituitary facilitates the progression of primary to dominant preovulatory follicles ( Yoshino et al., 2011 ). Additionally, FSH causes the breakdown of LDs in porcine GCs ( Liu et al., 2021 ), leading to the synthesis of progesterone via the Beclin1 protein. When porcine GCs were exposed to ATG5siRNA, promoting autophagosome formation, or CQ, an autophagy inhibitor, there was a notable decrease in FSH-induced progesterone production. This indicates the critical role of autophagy in progesterone synthesis within ovaries. Recent findings ( Shao et al., 2022 ) underscore the importance of the gene regulator WT1 in follicle formation. Overexpression of WT1 affects normal granulosa cell development, while heterozygous mutations in WT1 result in subfertility in female mice, accompanied by reduced expressions of the FSH receptor and cytochrome P450 family 19 subfamily A member 1, commonly referred to as aromatase ( Gao et al., 2014 ). Inhibiting autophagy leashes to a buildup of WT1 protein in GCs, diminishing the levels of receptor and protein, thereby disrupting GC differentiation ( Galano et al., 2022 ). To modulate WT1 levels, Epg5 facilitates WT1 breakdown via p62 in GCs. Mice with Epg5 deletions display a phenotype akin to individuals with premature ovarian failure ( Liu et al., 2023 ). Aging is linked with increased apoptosis and senescent cells. In the absence of Epg5, GCs in the ovary retain WT1, which would typically be degraded during folliculogenesis from secondary to antral follicles, resulting in subfertility in mice. Conversely, during folliculogenesis—where differentiation is not reliant on FSH—bone morphogenetic protein-2 (BMP2) improves GC proliferation through sphingosine kinase-1 ( Ito et al., 2021 ). Considering that WT1 modulates the expression of BMG2 ( Gao et al., 2014 ), it would be plausible that autophagy influences the entire spectrum of folliculogenesis in the ovaries, from early to late stages. As it is a fact that steroidogenesis is a complex process which involves several enzymes and regulatory factors with pivotal roles from cholesterol transport to the synthesis of specific hormones within different cell types ( Shoorei et al., 2023 ), thereby making it crucial for the production of key sex steroids. The interplay between hormones, receptors, and enzymes like CYP450arom highlights the intricate regulatory mechanisms governing steroidogenesis ( De Pascali et al., 2018 ; Widhiantara et al., 2021 ). Several studies have reported variations in sex hormone synthesis upon bisphenol A exposure. For instance, when rat offsprings prenatally exposed to bisphenol A it disrupted steroid production ( Nguyen et al., 2020 ). Higher bisphenol A doses of 40 mg kg −1 made the pregnant rats vulnerable to abortion and significant reduction of pups survival ( Wei et al., 2020 ). A study on freshwater fish, Gobiocypris rarus, showed DNA and histone methylation in the ovaries and long term and short term exposure lead reduced steroid hormones ( Liu Y. et al., 2020 ). In short, autophagy is vital for ovarian physiology, influencing folliculogenesis, ovulation, and luteal development, impacting germ cell survival and fertility. Age-related decline correlates with increased oxidative stress and reduced fertility in older women, while autophagy plays a dual role in CL dynamics and modulates progesterone synthesis. Additionally, understanding the impact of endocrine disruptors on autophagy-mediated steroidogenesis in ovarian cells is essential for comprehending their broader effects on hormonal balance. Genomic studies underscore its significance in sexual and follicular development, offering insights into reproductive health.

The complex interplay between autophagy and steroidogenesis, particularly in the endocrine system, reveals a fascinating landscape of cellular mechanisms and pathways. Autophagy, a fundamental cellular process, has been recognized as a critical player in maintaining cellular homeostasis, eliminating damaged components, and facilitating various physiological functions. Its role in the context of steroid hormone production, especially testosterone and progesterone, underscores its significance in reproductive health and endocrine regulation. The research elucidates the nuanced role of autophagy in Leydig and GCs, pivotal players in testicular and ovarian steroidogenesis, respectively. From the degradation of organelles essential for hormone production to modulating the expression of crucial proteins involved in steroid synthesis, autophagy emerges as an important regulator. The autophagy modulation, as observed in various studies, directly correlates with changes in steroid production rates, indicating a tightly regulated relationship. Furthermore, insights into the regulatory mechanisms of autophagy, such as the involvement of specific genes, signaling pathways, and external factors, provide a comprehensive view of its orchestration within the endocrine system. The intricate balance between autophagy and steroidogenesis has profound implications for understanding fertility, aging-related endocrine disorders, and potential therapeutic interventions. Given the significant impact of autophagy modulation on steroid production, future research may focus on developing targeted therapeutic interventions. Drugs that selectively enhance or inhibit autophagy in specific cells could offer novel treatments for conditions like hypogonadism, infertility, or age-related endocrine disorders. In addition, delving deeper into the molecular mechanisms underlying the interplay between autophagy and steroidogenesis will be important. Identifying additional genes, pathways, or molecules that regulate this relationship could provide more precise targets for therapeutic interventions. While some studies have explored the effects of EDCs like bisphenol AF and methyl tert-butyl ether on Leydig cell function and hormone production, there’s still much to learn about how they affect different types of cells and hormonal pathways. Understanding how EDCs affect hormone production could help us identify ways to protect reproductive health. It is also crucial to understand the underlying molecular mechanisms behind changes in hormone levels seen with exposure to EDCs like bisphenol A. This knowledge could lead to new ways to prevent or treat hormone-related health issues. Extending research from animal models to human studies will be pivotal. Investigating the role of autophagy in human Leydig and GCs, especially in pathological conditions like endometriosis, polycystic ovary syndrome, or testicular disorders, will provide invaluable clinical insights. Furthermore, given the age-related decline in autophagy and its implications for endocrine health, further research into rejuvenating autophagic processes in aging cells or tissues could have profound implications for extending reproductive health and overall longevity. To sum up, the intricate relationship between autophagy and steroidogenesis within the endocrine system represents a burgeoning field of research with vast clinical implications. By unraveling the complexities of this relationship, scientists and clinicians alike stand poised to revolutionize treatments for a myriad of endocrine disorders, ultimately enhancing reproductive health and quality of life.

Autophagy is referred to as an intracellular catabolic pathway that is genetically determined and evolutionarily conserved from yeast to higher primates ( Vargas et al., 2023 ). In typical physiological contexts, autophagy mediates the internalization of cellular cargoes such as; old protein and damaged organelles with lysosomes thereby sequestering and reutilizing to maintain cellular homeostasis ( Debnath et al., 2023 ). Lysosomal degradation further characterizes a common endpoint of various autophagic mechanisms, such as chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy, hither ahead denoted as autophagy; each with distinct purpose and indiscriminate sequestration and degradation ( Levine and Klionsky, 2004 ; Ma et al., 2023 ). Additionally, cells engage other specialized mechanisms for selective targeting, such as lipophagy, zymophagy, mitophagy, and crinophagy; to target specific substrates ( Vargas et al., 2023 ). The former ones are usually referred to as “bulk” or “non-selective” autophagy and the latter ones are denoted as “selective” autophagy. While the existence of autophagy has been recognized for some time, its comprehensive exploration has only recently gained momentum. Besides its homeostatic functions, autophagy significantly influences the disease course of almost all cancers, various infections, immune responses, and multi-organ disorders, as well as neurodegenerative conditions ( Nanayakkara et al., 2023 ; Yamamoto et al., 2023 ). Autophagy works as a quality-control process, removing invading pathogens, protein masses, and dysfunctional or senescent proteins and organelles from cells ( Yao et al., 2021 ). Additionally, it plays important roles in diverse processes such as cellular differentiation, embryonic development, and aging, potentially offering protective effects ( Wang et al., 2019 ; Aman et al., 2021 ). The recognition of autophagy’s substantial implications in various diseases has heightened research interest in exploring its physiological and pathological aspects. Earlier, we have discussed autophagy in relation to lipolysis ( Khawar et al., 2019 ; Khawar et al., 2021a ) in the liver ( Nazeer et al., 2023 ) and reproduction ( Gao et al., 2019 ; Gao et al., 2020 ; Khawar et al., 2022 ). Interestingly, extensive research on autophagy in normative and pathological endocrine settings has yielded promising knowledge, whereby a unified understanding of steroidogenesis in reproductive organs remains enigmatic. Despite the promising implications of autophagy (as summarized in Table 1 ) in preclinical investigations, and as of our current knowledge, there exists a notable literature gap and further elucidation of these mechanistic insights is imperative to advance our comprehension and potentially pave the way for novel therapeutic interventions in reproductive health. Therefore, we, herein, explore the involvement of autophagy in ovarian and testicular steroidogenesis, along with the scrutiny of its regulatory mechanisms. Summary of recent studies investigating autophagy in testis and ovaries. AFB1, aflatoxin B1; CIRBP, cold-induced RNA-binding protein; FSH, follicle stimulating hormone; HFD-HF, high-fat-high fructose diet; NP, nonylphenol, PCOS, polycystic ovary syndrome, RA: retinoic acid, ROS: reactive oxygen species.