Functions of Lysosomes in Mammalian Female Reproductive System

Abstract

Introduction The lysosome was first described and named by the Belgian cytologist and biochemist De Duve et al. in 1955.[1] It is the most acidic membrane-bound intracellular organelle, with a lumen pH of ˜4.5-5.0. This environment is optimal for >60 different hydrolytic enzymes in the lysosome, such as proteases (e.g., cathepsins), lipases, nucleases, glycosidases and phosphatases, that can break down biomolecules within intracellular and extracellular origins. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) to pump H+ into the lysosomal lumen and counter ion channels to dissipate the transmembrane voltage built up by V-ATPase.[2] In addition to its digestive role, the lysosome is also important for intracellular trafficking, cellular homeostasis, metabolic signaling, cholesterol transport, lipid metabolism, immune response, and hormonal signaling, etc.[2-6] Mutations in lysosomal genes could potentially lead to lysosomal dysfunction. Disrupted lysosomal functions in degradation, export, or trafficking can lead to abnormal accumulation of lysosomal materials, resulting in >50 rare inherited metabolic disorders in humans, collectively termed lysosomal storage diseases.[7] The lysosome participates in essential cellular processes such as endocytosis and exocytosis [Figure 1], autophagy [Figure 2], and cell death [Figure 3].Figure 1:: Four main types of endocytosis and a proposed exocytosis involving lysosomes. (A) Clatherin-mediated endocytosis. (B) Clatherin-independent endocytosis. (C) Pinocytosis. (D) Phagocytosis. (E) A few events involving endocytosis in the female reproductive system. (F) A proposed lysosomal exocytosis. (G) A few events potentially involving exocytosis in the oviduct and uterus. Part of the figure was created using BioRender.Figure 2:: Three types of autophagy. (A) Macroautophagy. (B) Chaperone-mediated autophagy. (C) Microautophagy. (D) A few events involving autophagy in the female reproductive system. Part of the figure was created using BioRender.Figure 3:: The lysosome and cell death. (A) Involvement of lysosomes in different types of cell death, including lysosome-dependent cell death. (B) A few events involving lysosome-dependent cell death in female reproduction. Part of the figure was created using BioRender.Endocytosis transports extracellular cargo molecules to the lysosome for processing. There are four main types of endocytosis [Figure 1A-1D]: clathrin-mediated endocytosis,[8] clathrin-independent endocytosis,[9] pinocytosis,[10] and phagocytosis.[11] The involvement of lysosomes in exocytosis is relatively less studied and the mechanisms involved are still under investigation. Lysosomes and lysosome-related organelles can be transported to and fused with the plasma membrane to release the lysosomal contents into the extracellular space. Increased intracellular calcium level and depletion of cholesterol can both trigger lysosomal exocytosis, which is a tightly regulated process involving microtubules and actins, Rab GTPases, and SNAREs,[12] [Figure 1F]. Autophagy delivers unwanted intracellular cargo molecules, which could be of extracellular origin, to the lysosome for degradation and nutrient cycling.[13] There are three main types of autophagy [Figure 2A-2C]: macroautophagy, chaperone-mediated autophagy, and microautophagy. The primary type of autophagy is macroautophagy. It requires the formation of autophagosomes [Figure 2A] that involves LC3/MAP1LC3 (microtubule-associated proteins 1A/1B light chain 3B, cytosolic form LC3-I, membrane form LC3-II). Autophagosomes fuse with lysosomes to form autolysosomes for cargo degradation by lysosomal hydrolases. Many genes, such as autophagy-related (ATG) genes, have been identified to play essential roles in the autophagic pathways and implicated in the functions of the female reproductive tract.[13] There are generally two types of cell death, regulated cell death and accidental cell death. Lysosome-dependent cell death [Figure 3] is a type of regulated cell death,[14] in which lysosomal membrane permeabilization causes selective release of cathepsins or massive release of lysosomal enzymes leading to cell death. It could also trigger other types of regulated cell death, such as apoptosis and pyroptosis, which is a highly inflammatory form of programmed cell death in which cathepsins are involved.[15] The mammalian female reproductive system generally consists of two ovaries, two fallopian tubes/oviducts, a uterus/two uterine horns, a placenta/placentas, a cervix, a vagina, and mammary glands. During the years following the discovery of the lysosome, studies dating back to 1973 revealed the presence of lysosomes and/or the activities of lysosomal enzymes in the ovary, uterus, cervix, and vagina using electron microscopy and biochemical approaches. Many of these studies were previously reviewed.[16,17] Since then, more studies in different species have implicated lysosomes in the female reproductive system, but have not been comprehensively reviewed. This review updates the research on the functions and mechanisms of lysosomes in the female reproductive system. It will be organized roughly based on the order of female reproductive organs involved in the pregnancy process: ovary, oviduct, uterus, placenta, parturition, cervix and vagina, and mammary gland [Figure 4].Figure 4:: Summary of known or suggested functions of lysosomes in the female reproductive tract. (A) Fallopian tube (oviduct). (B) Uterus. (C) Placenta. (D) Vagina. (E) Mammary gland. Part of the figure was created using BioRender.Functions of Lysosomes in the Ovary The ovary is an essential part of the hypothalamic-pituitaryovarian (HPO) axis connecting the central nervous system and the female reproductive system [Figure 5]. The ovary contains primary oocytes, developing follicles, atretic follicles, interstitium, and corpus luteum (corpora lutea, plural). It is the site for oocyte maturation. Although lysosomes do not seem to have a substantial presence in the oocyte, they play an essential role in follicle atresia, ovulation, luteal regression, luteal cell survival, and ovarian steroidogenesis [Figure 6].Figure 5:: Functions of lysosomes in the HPO axis and steroidogenesis. (A) Female reproductive system, including the ovary, fallopian tube/oviduct, uterus, placenta (not shown), cervix, and vagina. The female reproductive system is under the control of HPO axis. After puberty, the releasing of GnRH from hypothalamus will bind to GnRH receptor on anterior pituitary to stimulate the secretion of FSH and LH. During follicular phase, the FSH binds to FSH receptor on granulosa cells to induce estrogen (E2) synthesis. Meanwhile, LH binds to LH receptor on theca cells to induce the synthesis of androgens, which will be transferred to granulosa cells for E2 synthesis. E2 will have negative feedback on LH and FSH secretion (B). During follicular phase, E2 causes proliferating effects on the endometrium of the uterus (A). During pre-ovulation, on the other hand, E2 will accumulate and have positive feedback on the LH and FSH secretion, which will lead to ovulation (C). After ovulation, the granulosa cells and theca cells will differentiate into lutein cells in the corpus luteum. Similar as in the follicle phase, granulosa lutein cells and theca lutein cells also receive LH and FSH signaling from the anterior pituitary. The main difference is the high level production of progesterone (P4), which is stimulated by LH acting on both granulosa and theca lutein cells. P4 and E2 will send negative feedback for LH and FSH secretion (D). The combination of E2 and P4 from corpus luteum will induce secretory effects on the endometrium of the uterus (A). Lysosomes (pink vesicles) are shown to regulate steroidogenesis in the preparation of free cholesterol, the substrate for steroidogenesis, as well as in the degradation of LH and LHR and FSH and FSHR to control HPO axis in regulating steroidogenesis in the ovary. HPO: Hypothalamic-pituitary-ovarian; GnRH: Gonadotropin-releasing hormone; LH: Luteinizing hormone; FSH: Follicle-stimulating hormone; LHR: Luteinizing hormone receptor; FSHR: Follicle-stimulating hormone receptor; E2: Estradiol; P4: Progesterone. This figure was created using BioRender.Figure 6:: Functions of lysosomes during ovarian cycle. Ovarian cycle is a series of changes in the ovary, from oogonium, to primordial follicle and a few more stages, to Graafian follicle, and corpus luteum after ovulation. Most follicles will be removed before reaching Graafian follicles via follicular atresia. Only a few follicles can fully develop from primordial follicles to Graafian follicles, continue with ovulation and corpus luteum formation to go through the full ovarian cycle. Lysosomes have known or suggested functions in follicular atresia, ovulation, luteal maintenance and luteal regression, and steroidogenesis in the ovary. Part of the figure was created using BioRender.Lysosomes and hypothalamic-pituitary-ovarian axis The key hormones in the HPO axis include gonadotropin-releasing hormone (GnRH) from the hypothalamus, luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary, and estrogen and progesterone from the ovary [Figure 5]. Lysosomes were detected in the GnRH neurons of juvenile monkey hypothalamus.[18] Increased volume fraction of lysosomes/lipofuscin was detected in the GnRH neurons of aged female rats.[19] Lysosomes had morphological changes in response to LH-releasing hormone (LH-RH) injections[20] and were involved in the internalization of LH-RH antagonist in the LH gonadotrophs of female rats,[21] suggesting potential functions of lysosomes in regulating LH production. Studies have shown lysosomal responses, especially activities of lysosomal enzymes, to LH and FSH and their analogs in the ovary. LH and FSH can reduce lysosomal cathepsin D activity in rat granulosa cells of the preovulatory follicles.[22] Equine chorionic gonadotropin/pregnant mare serum gonadotropin (eCG/PMSG) can change the activities of acid phosphatase, N-acetyl-beta-D-glucosaminidase and beta-glucuronidase in the follicular fluid, granulosa and theca cells of preovulatory follicles of immature Wistar rats.[23] PMSG and human chorionic gonadotropin induced superovulation in immature rats is accompanied with increased acid phosphatase activity in granulosa and theca cells of Graafian follicles.[24] Lysosomes and ovarian follicle atresia Follicles are the functional units in the ovary. Most of them contain an oocyte surrounded by granulosa cells and in later stages also theca cells lining up the outer layer. They develop from primordial follicles to Graafian follicles (preovulatory follicles) prior to ovulation. In mammals, the majority of follicles are removed by atresia during follicle development. Follicular atresia is a hormonally controlled degenerative process initiated with the death of granulosa cells and ended with the degeneration of oocytes. It was indicated in ewes that the large atretic antral follicles showed more lysosomal rupture in granulosa cells than the small ones. Correspondingly, necrosis appeared to be more dominant in the granulosa cells of large atretic follicles, while apoptosis was more prevalent in the granulosa cells of small atretic follicles.[25] The role of lysosomes in follicular atresia is evolutionarily conserved as cathepsin D-mediated apoptosis in the clearance of follicular cells during follicular atresia is also observed in Nile tilapia.[26] Oocyte atresia involves autophagy and apoptosis. Since lysosomes are important for both events [Figures 2 and 3], it was not surprising to find a high number of active lysosomes and autophagolysosomes accompanied with increased activity of lysosomal enzymes in oocytes that were undergoing atresia. It is proposed that the degeneration of oocyte started with autophagy, which can then trigger the apoptosis pathways to degrade nuclear components and remove the cytoplasmic components.[27] Lysosomes and ovulation After follicle maturation, a few selected Graafian follicles will undergo ovulation to release the oocytes. Ovulation is a complexed process that involves neuroendocrine regulation, communications among multiple cell types, extensive extracellular matrix remodeling of follicular apex, loss of the surface epithelium, and eventually follicle rupture and oocyte release.[28] During ovulation, there was an increased concentration of lysosomal enzymes in the rat ovarian bursa fluid.[29] It was also observed that the lysosomes from the epithelium covering the Graafian follicles would aggregate into groups and fuse with each other right before ovulation in the rabbit. They would move to the plasma membrane and release the lysosomal enzymes into the extracellular space expectedly to assist follicle rupture during ovulation.[30] These observations suggest the involvement of lysosomes in ovulation. Lysosomes and luteal regression Upon ovulation, the oocyte with cumulus cells are released from the mature follicle, while the granulosa and theca cells in the remaining ruptured follicle will differentiate into luteal cells to form a corpus luteum.[31] The corpus luteum is a temporary endocrine structure that is the main site for progesterone production during early pregnancy in some species, and throughout the pregnancy for other species. Progesterone is an essential hormone to support pregnancy. If pregnancy does not occur or another organ takes over progesterone production during pregnancy, the corpus luteum will undergo luteal regression/luteolysis. Luteal regression is associated with increased number and size of lysosomes, as well as increased activities of lysosomal enzymes in bovine, porcine, and mouse corpora lutea.[32-34] Autophagy, apoptosis, and necrosis have been suggested as mechanisms for lysosomal involvement in corpus luteum regression.[32,35] Prostaglandin F2alpha (PGF2α), an inducer of luteal regression in domestic animals, can increase the release and activities of lysosomal enzymes, therefore accelerate the regression of corpus luteum.[36] It is not surprising that a KEGG pathway analysis of primate luteal transcriptome revealed differential expression of genes in “lysosome” category between rescued and regressing corpora lutea.[37] Lysosomes and luteal cell survival Although it may seem to contradict the role of lysosomes in luteal regression, our recent study suggests a novel role of lysosomes in luteal cell survival.[38] TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1, encoded by mucolipin 1 (MCOLN1, Mcoln1)) is an important lysosomal counter ion channel. Mutations in the mucolipin 1 gene can lead to a lysosomal storage disorder called MLIV (Mucolipidosis Type IV) in both humans and mice, which share common clinical features, such as impaired vision, paralysis, and elevated plasma gastrin.[2,39] MLIV patients do not reproduce[40] but the mechanism of their infertility is unknown. TRPML1 is highly expressed in mouse luteal cells. TRPML1 deficiency in mice led to infertility in 5-6 months old Mcoln1-/- female mice, accompanying with progesterone deficiency and luteal cell degeneration during early pregnancy.[38] Because TRPML1 is required for efficient fusion of autophagosomes with lysosomes, TRPML1 deficiency leads to impaired fusion of autophagosomes with lysosomes for degradation and subsequent accumulation of autophagosomes, which could lead to degenerative cell death in Mcoln1-/- mice and MLIV patients.[39] Extensive luteal cell vacuolization in the Mcoln1-/- corpus luteum most likely represented autophagic vacuoles that were also observed in the Mcoln1-/- gastric epithelial cells[39] and possibly represented spaces of accumulated lipid droplets as well.[38] Since TRPML1 deficiency is related to mitochondrial fragmentation that could contribute to progressive cell degeneration, and there is reduction of mitochondrial marker HSP60 in Mcoln1-/- corpora lutea, Mcoln1-/- luteal cell degeneration could be contributed by both defective lysosomes and defective mitochondria.[38] Because of the essential role of TRPML1 for lysosomal functions, this study in the Mcoln1-/- mice suggests a novel role of lysosomes in luteal cell survival.[38] Lysosomes and ovarian steroidogenesis Lysosomes have multiple roles in ovarian steroidogenesis, which is controlled by the HPO axis [Figures 5 and 6]. The steroid hormones are produced from the common precursor, cholesterol. The main source of cholesterol is through endocytosis of cholesterol rich low-density lipoprotein (LDL) or selective uptake of cholesterol esters from high-density lipoprotein and a minor source is through de novo cholesterol synthesis.[41] A major role of lysosomes in steroidogenesis is to liberate LDL-embedded cholesterol derived from endocytosis.[42] Lysosomal liberation of cholesterol involves lysosomal enzymes and two Niemann-Pick Type C (NPC) proteins: NPC1, a lysosomal membrane protein responsible for exporting free cholesterol, and NPC2, a small and soluble protein binding to cholesterol in the lysosome lumen. NPC1 deficiency leads to female infertility with impaired ovarian function, including impaired ovarian steroidogenesis, caused by defective hypothalamic control of the pituitary in the HPO axis.[43] NPC2 is mainly detected in the theca cells of large and healthy antral follicles, as well as in the luteal cells of the corpora lutea. NPC2 deficient female mice were infertile as they failed to ovulate. They had normal LH and FSH levels but reduced serum estrogen level and increased ovarian cholesterol level, suggesting defective cholesterol export from intracellular stores in the ovarian steroidogenic cells.[44] Lysosomes are also involved in the degradation of regulators of steroidogenesis in the ovary, such as LH-LHR[45] and FSH-FSHR complexes,[46] and intrinsic receptors for PGF2α.[47] In addition to roles in the preparation and transport of the substrate cholesterol and degradation of regulators of steroidogenesis, lysosomes may also have a role in luteal cell survival for steroidogenesis in the corpus luteum.[38] Functions of Lysosomes in the Oviduct The oviduct (or fallopian tube in humans) consists of four segments: infundibulum, ampulla, isthmus, and utero-tubal junction. Early pregnancy events, including acrosomal exocytosis, fertilization, early embryo development, and embryo transport towards the uterus, occur in the oviduct. Studies have shown the presence of lysosomes and lysosomal enzymes in the oviduct. Many primary lysosomes and secondary lysosomes were detected in the nonciliated epithelial cells of sheep oviductal isthmus during early pregnancy.[48] Lysosomes in the oviductal epithelial cells, mainly at the preampulla segment, are involved in the uptake of serum immunoglobulins or intravenously injected tracers (e.g., horseradish peroxidase [HRP] and ferritin) via basolateral endocytosis, and possibly in the digestion of the cargoes (e.g., tracers) during mouse pregnancy.[49,50] The injected tracers were also detected in apical vesicles of the mouse oviductal epithelial cells, most likely for releasing into the oviductal lumen via exocytosis, in which the role of lysosomes was previously unknown.[50] A proteomics study revealed several lysosomal enzymes in the bovine oviductal fluid, suggesting the participation of lysosomes in the apical secretion of oviductal epithelial cells.[51] The functions of lysosomal enzymes in the oviductal lumen remain to be investigated. Lysosomal enzyme uteroferrin, which is an iron-containing lysosomal acid phosphatase, was detected in the ampulla and isthmus epithelial cells of both cycling and pregnant pigs. Since uteroferrin did not seem to be secreted into the oviductal lumen but uterine lumen,[52] it suggested different vesicle trafficking systems and/or different roles of uteroferrin in the oviduct and uterus. These observations suggest general roles of lysosomes in oviductal epithelium for vesicle trafficking and nutrient recycling. Functions of Lysosomes in the Uterus The uterus is the only viable place for a mammalian embryo/fetus to grow to term. The uterine endometrium includes uterine luminal epithelium (LE), glandular epithelium, stromal cells, endothelial cells, and immune cells. It is under the control of ovarian hormones estrogen and progesterone, and undergoes dynamic changes during the menstrual cycle/estrous cycle and pregnancy. Lysosomes have been implicated in the dynamic changes in the endometrium. Lysosomes and cycling uterus A female menstrual cycle includes: proliferative phase, secretory phase, and menstruation. Early studies revealed increased lysosomal activities in the glandular epithelium during secretory phase, which may contribute to the enhanced secretion from glandular epithelium during secretory phase and imply a role of lysosomes in exocytosis. Macrophages are phagocytic immune cells that play important roles in the uterus during menstrual/estrous cycle and pregnancy.[53] They are enriched with lysosomal enzymes, such as acid hydrolases. They disappeared in the secretory endometrium and reappeared upon the onset of menstruation, most likely to provide their phagocytic function in which the lysosomes are necessarily involved.[17] Progressively increased autophagy and apoptosis in human endometrium from early proliferative phase to late secretory phase were shown by the expression of MAP1LC3A-II and cleaved caspase 3, respectively.[54] This study further demonstrated the involvement of lysosomes in autophagy and apoptosis in cultured human endometrial cells.[54] These observations imply cell type specific roles of lysosomes in the human endometrium during menstrual cycle. Lysosomes and lysosomal enzymes in the uterus are responsive to different treatments. The lysosomes in the uteri of Wistar rats can sequester aluminum nitrate and indium sulfate, implying lysosomal function in the defense of xenobiotics.[55] PMSG treatment in immature Wistar rats induced the activities of beta-glucosaminidase and acid phosphatase activities but not that of beta-glucuronidase in the endometrium.[23] Progesterone treatment in ovariectomized rabbits upregulated the activities of a few lysosomal hydrolases, including acid and alkaline phosphatases and beta-galactosidase, in the endometrium.[56] These observations suggest lysosomal functions in uterine defense of xenobiotics and uterine response to hormones. Lysosomes and uterine endocytosis and secretion The uterus and its enclosed uterine lumen undergo dynamitic changes in sizes and contents. Endocytosis and exocytosis play important roles in these dynamics[57] and the lysosome has functions in both processes [Figure 1]. Lysosomes are involved in the temporal and directional changes of endocytosis in the mouse uterine epithelium during early pregnancy. On both 0.5 days postcoitus (D0.5) and D4.5, 2 h after intravenous injection, the injected HRP tracer was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies and elongated dense bodies (both of which were positive with lysosomal acid phosphatase), and many small vesicles near the apical surface of the cells. The HRP may be excreted into the uterine lumen and/or digested in the lysosomes. On the other hand, when the HRP was injected into the uterine lumen on these days for 20-40 min, there was no detectable uptake of HRP from the lumen into LE or glandular epithelium on D0.5, but a large amount of HRP was taken up from the uterine lumen into apical endocytic vesicles, multivesicular bodies and dense bodies (lysosomes) in both LE and glandular epithelium on D4.5[58] [Figure 7]. Similar as in mice, LE endocytosis of tracers from uterine lumen during peri-implantation was also observed in rats (using HRP or ferritin as tracers)[59] and rabbits (using labeled uteroglobin as a tracer).[60] Extensive early studies demonstrated that LE apical endocytosis, indicated by the uptake of tracer(s) injected into the uterine lumen, was upregulated by progesterone in both mouse and rat, and peaked around the time of the establishment of uterine receptivity for embryo implantation.[59]Figure 7:: Proposed functions of lysosomes in endocytosis and exocytosis of uterine LE during early pregnancy in mice. (A) On 0.5 days postcoitus (D0.5). Based on (Tung et al. 1988), 2 h after intravenous injection of a tracer HRP, HRP was detected in the endocytic vesicles along the basolateral membrane, multivesicular bodies, and lysosomes, as well as vesicles near the LE apical surface. HRP could be degraded in lysosomes or released to the uterine lumen. Endocytosis of HRP injected into the uterine lumen did not occur in the LE on D0.5. (B) On D4.5 (˜D4 12 h). It is ˜ 12 h after embryo attachment and ˜ 8 h before trophoblast penetration through the LE in mice. Increased endocytosis of intravenous injected HRP was detected in the basolateral LE compared to D0.5. HRP injected into the uterine lumen could be detected in the LE vesicles, indicative of apical endocytosis on D4.5 (Tung et al. 1988). LE vesicle trafficking could deliver messages from the maternal side to the embryo, and vice versa. Extracellular vesicles, including exosomes derived from multivesicular bodies in LE and trophoblasts, are newly recognized players in the maternal-embryo communications that had been demonstrated in sheep.[ 71 ] Part of the figure was created using BioRender. HRP: Horseradish peroxidase; LE: Luminal epithelium.Lysosomal enzymes can be secreted into uterine lumen under certain circumstances. One example is uteroferrin, a lysosomal acid phosphatase. It is upregulated by progesterone and secreted by uterine epithelium during estrous cycle and pregnancy in pig.[52] Although uteroferrin is a lysosomal enzyme, its secretion into uterine lumen may not involve the lysosome per se because it could be secreted before being targeted to the lysosomes.[61] It was suggested that uteroferrin may carry/transport iron from the uterus to the conceptus for fetal development.[52,61] Lysosomal enzymes have dynamic activities in the uterine lavage fluid of nonparturient, postpartum, and ovariectomized postpartum mares.[62] In nonparturient uterine lavage fluid, acid phosphatase, beta-glucuronidase (B-Gase), and N-acetyl-beta-D-glucosaminidase (NAGase) had increased activities in mid-and late-diestrus than in mid- to late-estrus. In 1-4 days postpartum uterine lavage fluid, the activities of acid phosphatase, B-Gase, and NAGase were high but declined rapidly thereafter. These activities were progesterone-dependent. They tended to be higher in the uterine lavage fluid of intact mares than in ovariectomized ones on day 16 postpartum.[62] These findings indicate dynamic endometrial lysosomal secretory activities, which are under hormonal control and are likely associated with endometrial remodeling during estrous cycle and postpartum. Uterine epithelial acidification upon embryo implantation An early study implied a more acidic intralysosomal pH in the rat uterine epithelium upon embryo implantation.[63] When nonspecific acid phosphatase activities towards substrates beta-glycerophosphate and p-nitrophenyl phosphate at both pH = 5.0 and pH = 6.0 were evaluated in rat uterine epithelium, it was found that at late diestrus and D5.5 (post-implantation initiation), a significantly larger lysosomal population in the LE and glandular epithelium was operating at pH = 5.0 rather than pH = 6.0; while at estrus, the epithelial lysosomes showed no preference for an operational pH = 5.0 or 6.0. In addition, LE lysosomes had a substrate preference for p-nitrophenyl phosphate over beta-glycerophosphate on D5.5 but not at estrus or late diestrus.[63] D5.5 LE also had increased lysosomal population and more active lysosomes with frequent invaginated or vesiculated forms compared to LE of nonpregnant rats.[63] These observations suggest dynamic changes, including intralysosomal pH, in LE lysosomes upon embryo implantation. Our microarray analysis of the peri-implantation mouse LE revealed dramatic upregulation of Atpase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2),[64] a gene encoding a tissue-specific d subunit of V-ATPase. V-ATPase regulates extracellular lumen pH (when it is located on plasma membrane) and pH in acidic intracellular organelles (when it is located on intracellular organelle membrane), such as lysosomes. This microarray study led us to the novel finding of uterine epithelial acidification during implantation initiation,[65] detected by LysoSensor Green DND-189 (pKa ˜5.2). Since the lysosome is the most acidic intracellular organelles within the pH range (pH <5) detectable by LysoSensor Green DND-189, the uterine epithelial acidification detected by LysoSensor Green DND-189 indicates lysosomal acidification, although acidification of other intracellular organelles couldn't be ruled out. Uterine epithelial acidification also occurred in the artificially induced decidualization mouse uterus,[65] indicating the involvement of maternal factors in regulating this process. We further demonstrated that uterine epithelial acidification was associated with uterine receptivity: V-ATPase inhibitor bafilomycin A1 suppressed/delayed uterine epithelial acidification and embryo implantation in a natural pregnancy mouse model as well as uterine preparation for embryo implantation in an artificially induced decidualization mouse model using uterine fat pad injection.[65] However, the detailed mechanisms of how this acidification affects implantation require further investigation. Lysosomes and embryo implantation Successful embryo implantation requires synchronized com