Relevance of lipid homeostasis to male infertility

Spermatogenesis marks dynamic changes in germ cell lipidome

The developing male germ cells undergo drastic changes in their lipid content during spermatogenesis.^[14] As sperm lack membrane-bound organelles and relies heavily on the lipid content in their plasma, acrosomal, and nuclear membranes for proper function, amidst spermatogenesis, extensive membrane-lipid changes occur during meiotic division, spermiogenesis, epididymal maturation, and finally before fertilization.^[15] Moreover, studies have reported that failure of these modulations adversely impacts male fertility.^[16-18]

During meiosis, the germ cells encounter a shift in polyunsaturated fatty acids (PUFAs) content to facilitate membrane fluidity and stability during the cell division process. While along spermiogenesis, there is a significant enrichment in docosapentaenoic acid, which confers proper membrane characteristics to mature sperms, essential for their motility, membrane fluidity, and ability to fuse with the oocyte.^[16] These changes occur under controlled lipid homeostasis, as an increase in saturated fatty acids (SFAs) inhibits PUFA synthesis.^[19]

Although sperms lack active protein synthesis, post-testicular sperm maturation occurs in the epididymis through lipid-rich exosomes, known as epididymosomes.^[20] A study reports that in rams, during epididymal maturation, enrichment in omega-3 fatty acids, particularly docosahexaenoic acid (DHA), occurs in sperms, reducing arachidonic acid, which ensures further increase in membrane fluidity and flexibility, needed for capacitation and fertilization process [Table 1].^[17] Studies report that the epididymosomes carry a diverse set of proteins, non-coding ribonucleic acids (RNAs) (ncRNAs), and distinct lipids, which they transfer to the sperm during epididymal storage. The proteins (enzymes and chaperones) help in post translational modification (PTM) of sperm proteins while ncRNAs transmit information, and importantly, the lipids help information of membrane lipid rafts, which play a vital role in sperm capacitation and acrosome reaction (AR).^[21] Interestingly, specific epididymosomes vary in lipid composition based on their location in the epididymal segment, which indicates they probably modulate sperm membrane fluidity by donating the cargo they carry.

As sperms ascend the female reproductive tract, further membrane lipid remodeling occurs, such as efflux of cholesterol and loss of phospholipids containing SFA tail, leading to extra increase in membrane fluidity, which signals the onset of capacitation.^[22] Essentially, physiological ROS promotes cholesterol oxidation at the sperm membrane surface, forming oxysterols that bind to extracellular albumin (hydrophilicity) and release cholesterol.^[23] This leads to hydrolysis of some phospholipids to lysophospholipids and fatty acids, which act as signaling factors promoting sperm motility, viability, and AR.^[24] Cholesterol depletion also redistributes lipid rafts on the sperm membrane, facilitating sperm receptor organization for sperm-oocyte interaction.^[25] Taken together, the above findings demonstrate the pivotal role of dynamic lipid profile in the development and maturation of sperm.

Lipid composition of the sperm cells and their function

Sperm membranes have distinct features crucial for their physiological functions. Primarily, the membrane lipid content varies between the head and tail, with the tail having more lipid than the sperm head. Particularly, DHA and ceramides are present in the tail membrane that promotes sperm motility, while head lipid contains sphingomyelin (SM) and phosphatidylcholines (PCs) vital for the fertilization process. Besides sperm head membrane also exhibits lateral heterogeneity, meaning there are distinct regions with different lipid and protein compositions, which support raft formation and reorganization during AR and binding to the zona pellucida.^[24] Thus, the lipid content of sperm membrane is highly specialized, though considerable diversity exists among species. In general, the sperm membrane contains about 70% phospholipids, 25% neutral lipids, and 5% glycolipids.^[24]

Phospholipids are the major lipid component in sperm plasma membrane that includes glycerophospholipids (GPLs) and SMs. The GPLs are diacyl lipids on a glycerol backbone with a polar molecule. They include PC, phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), which contribute to sperm motility, lipid raft reorganization, membrane integrity, and cell signaling, respectively.^[26,27] In GPLs, SFAs are esterified at the sn-1 position while PUFAs are esterified at sn-2.^[27] PUFAs promote sperm membrane fluidity as unsaturated acyl chains weaken interaction with cholesterol, while SFAs increase membrane rigidity, that is also needed for membrane stability and integrity. However, both render sperm susceptible to ROS-driven oxidative damage [Table 1].^[28] Ether phospholipids or plasmalogens (another subclass of GPLs) are present abundantly on the anterior head region of sperm cell membrane, which enhances membrane fluidity and stability, as ether linkages do not get readily cleaved by lipase action.^[29] Cardiolipin, present abundantly in the sperm tail, maintains mitochondrial health needed for energy production and sperm motility.^[30] Lysophospholipids act as a signaling molecule for AR.^[24] SMs have a sphingosine backbone. In mammalian sperm, the head SMs predominantly contain very long-chain PUFAs that promote microdomain formation and fusion, influencing membrane structure. The tail SMs contain SFAs, which regulate flagellar movement in sperm cells.^[31]

Neutral lipids, the second major lipid component of the sperm plasma membrane, include cholesterol, diacylglycerol (DAG), and small amounts of cholesterol sulfate and cholesteryl esters. Cholesterol plays a key role in stabilizing the membrane by interacting with phospholipid, thereby regulating membrane stability.^[26] Importantly, cholesterol also contributes to the formation of membrane lipid rafts, maintaining their solidity, and also facilitates signal transduction.^[22] DAGs, minimally present in other cell membranes, are abundantly found in sperm plasma membrane, which enhances membrane fusion during AR through intra-acrosomal Ca2⁺ influx, which triggers acrosomal exocytosis.^[48,49]

Glycolipids, the third component of sperm plasma membrane, include sulfo-galactosyl-glycerolipid (SGG) or seminolipid, which primarily localizes in the sperm head, mediating binding of the sperm to the zona pellucida, and serves as an important biomarker for sperm fertilization potential.^[50] Importantly, cholesterol efflux alters glycosphingolipid conformation, exposing sugar residues that are recognized by lectins present in egg zona pellucida, aiding in sperm-egg interaction.^[22] Other glycolipids present in the outer leaflets of sperm plasma membranes promote membrane stability and intercellular communication.^[28]

Lipid metabolizing proteins related to sperm function and fertility

To investigate the effect of lipid-metabolizing proteins on male fertility, various knockout mice models have been developed. Male mice lacking key enzymes involved in phosphoinositide kinase (PIK3) signaling, ether lipid synthesis, sphingolipid biogenesis, or fatty acid desaturation exhibit impaired spermatogenesis with varying degrees of infertility.^[51-54] Phosphatidyl-inositol phosphates (e.g., PI4P, PIP2, PIP3) are membrane lipids involved in germ cell development and sperm motility. Deletion of p110 isoform of PIK3 led to embryonic lethality in most mice, and those that survived were infertile males.^[51] Similarly, knockout of Inositol polyphosphate 4-phosphatase II (INPP4B), a key enzyme in the PI signaling pathway, resulted in male mice with smaller testes and reduced sperm production.^[54] Other lipid metabolic gene knockouts also led to defective spermatogenesis and progressive sterility mentioned in Table 2. Besides, polymorphism in some lipid-metabolizing enzymes like cytochrome P450 (CYP), glutathione-Stransferase (GSTs), paraoxonase 1 (PON1), involved in detoxification and antioxidation, are reported to be associated with hyperlipidemia and male infertility. The single nucleotide polymorphism (SNP) variants – CYP1A1 (rs4646903), PON1 (rs662), and null alleles – GSTM1 and GSTT1 cause impaired detoxification that increases oxidative stress, resulting in a drastic decrease in sperm functions.^[55]

LDs: The main source for steroidogenesis

Unlike peptide hormones, steroid hormones are not stored in secretory vesicles but are synthesized and secreted into circulation immediately upon hormonal stimulation. This process requires readily available cholesterol, the precursor for steroid hormone synthesis. To meet this demand, steroidogenic cells such as LCs store cholesterol in the form of cholesterol esters (CE) together with triglycerides (TAG) into LDs, a fat storage organelle enclosed by a phospholipid monolayer embedded with proteins such as perilipins (PLIN) and cell death-inducing DFF45-like effector (CIDE).^[56-58] It has been shown that LD biogenesis occurs in a step-wise manner at specialized endoplasmic reticulum (ER) sites defined by functionally conserved ER membrane protein seipin (Sei1 in yeast).^[59] At these ER sites, seipin interacts with several factors, ensuring the fidelity of LD formation and preventing the detrimental effect of ectopic LD deposition in the ER.^[59] Strikingly, lack of seipin in steroidogenic tissues of mice, particularly in, testis, ovary, and adrenal glands, resulted in markedly reduced accumulation of CE-rich LDs, thus impairing production of steroid hormones.^[60] Primarily mobilization of lipid content from LDs is mediated by lipases that catalyze the breakdown of CE and TAG.^[56] However, recent studies have identified “lipophagy,” a form of selective autophagy, as a key mechanism in LD breakdown in steroidogenic cells, which promotes testosterone biosynthesis.^[61] In murine Leydig tumor cell line (MLTC) LC line and primary rat LCs, luteinizing hormone stimulation leads to dispersion of perinuclear LDs into smaller LDs, which associate with ER and mitochondria.^[62] Proteomic analysis has confirmed that these smaller LDs contain not only PLINs and CIDEs but also autophagosomal proteins, ER marker calnexin, and steroidogenic enzymes.^[62,63]

Several studies have demonstrated that lipophagy facilitates the hydrolysis of CE to release free cholesterol, which is then transported to mitochondria by steroidogenic acute regulatory (StAR) proteins, forming pregnenolone, which is further processed in the ER to produce testosterone.^[64,65] Another study in mice granulosa cells showed the presence of steroidogenic enzymes on the LD surface upon proteomic analysis. Thus, collectively these findings highlight the potent role of LDs as dynamic organelles critical for cholesterol storage and regulation of steroid hormone biosynthesis.^[66]

Dyslipidemia promotes ferroptosis: the main culprit for spermatogenic dysfunction

Dyslipidemia-induced oxidative stress triggers membrane lipid peroxidation, resulting in the accumulation of lipid hydroperoxides and other high reactive metabolites, which ultimately overwhelm the cellular antioxidant defenses.^[14] Oxidized PUFAs are cleaved from membrane phospholipids and catabolized through lipoxygenase enzymes (ALOX15, ACSL4, LPCAT3), generating a large amount of ROS, which attenuates antioxidizing factors (GPX, FSP1, and CoQ10), ultimately inducing ferroptosis - a form of cell death driven by iron-dependent lipid peroxidation.^[52] The resulting reactive radicals damage intracellular macromolecules, promote DNA fragmentation, and increase membrane permeability, leading to germ cell apoptosis, particularly arresting the round spermatids during spermatogenesis [Figure 2].^[14,52] Thus, in spermatogenic cells, derailed lipid homeostasis causes oxidative damage, and when this damage exceeds the cell’s threshold of peroxide tolerance, ferroptosis is initiated, leading to germ cell death. Notably, ferroptosis has been implicated as a characteristic feature of many male reproductive disorders, including heavy metal-induced testicular damage, testicular torsion, orchitis, hypogonadism, and even testicular cancers.^[68]

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Figure 2:

Ferroptosis causes testicular damage. Ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation, has emerged as a contributing factor in testicular damage. In testicular tissue, excessive iron accumulation paired with impaired antioxidant defenses – particularly reduced glutathione peroxidase 4 activity – leads to oxidative lipid damage. This oxidative stress disrupts the functional integrity of both Sertoli and Leydig cells, ultimately impairing spermatogenesis, suppressing testosterone synthesis, and triggering germ cell apoptosis. These cellular disturbances highlight ferroptosis as a potential link between oxidative stress and male infertility. GSH- Reduced glutathione, GSSG: Oxidised glutathione, GPx: Glutathione peroxidase, Fe²⁺: Ferrous ion, Fe³⁺: Ferric ion.

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Deregulated lipid homeostasis impairs the testicular microenvironment

In dyslipidemia, disrupted lipid metabolism significantly alters the testicular microenvironment and impairs cellular functions, compromising male fertility.^[68,69] Excess lipids alter the tight junction proteins, disrupting the blood-testis barrier and triggering testicular immune dysfunction.^[70] Conversely, Sertoli cells, which nourish developing sperms, suffer mitochondrial damage from abnormal lipid byproducts.^[69] Finally, increased reactive radicals exacerbate inflammation, damage cells and the intracellular components, activating testicular macrophages to clear apoptotic cells, inadvertently harming neighboring LCs.^[71-73] Overall, lipid imbalance in dyslipidemia severely impacts testicular somatic cells, leading to deterioration in steroidogenic and spermatogenic functions.

Dyslipidemia disrupts steroidogenesis

Dysregulated lipid metabolism hampers steroidogenesis through multiple mechanisms. Testosterone synthesis in LCs relies on cholesterol transport into mitochondria. However, elevated ROS in dyslipidemia oxidizes cholesterol to cholesterol hydroperoxide, and its co-trafficking into LC damages mitochondria, disrupting the redox balance, resulting in decreased testosterone production.^[56] Further, increased fatty acid and TAG levels suppress steroidogenic activity in LCs.^[57] A recent study has shown that culturing rat granulosa cells in high fatty acid media transformed the LD content from CE-rich core to TAG-rich core, reducing the release of free cholesterol needed for testosterone synthesis.^[74] Normally, lipophagy-mediated hydrolysis of CE-containing LDs into free cholesterol facilitates testosterone production, but, in hyperlipidemia, excess fatty acids inhibit lipophagy of LDs, further limiting cholesterol availability.^[64,73] In addition, another mechanism involves ROS-induced activation of adipocytes to generate leptin, which disrupts the hypothalamic-pituitary-gonadal (HPG) axis, resulting in low testosterone synthesis.^[75] Elevated leptin levels also inhibit the hedgehog signaling pathway required for the proliferation and maturation of Leydig stem cells.^[76]

Studies in animal models and humans

Studies indicate that individuals who are hyperlipidemic but not obese also experience compromised ovarian functions and increased risk of cardiovascular disease. This is primarily due to the lipotoxic effects of circulating LDLs imposed, which can cause cellular distortions.^[77] Several animal and human studies have explored the adverse impacts of dyslipidemia on male fertility [Table 3].

Rabbits, due to their sensitivity to dietary cholesterol and similarities with human lipid metabolism, serve as a valuable model to study hyperlipidemia-induced male infertility. In classical models, rabbits fed a diet with 2% cholesterol exhibited elevated plasma cholesterol levels and β-very low-density lipoprotein.^[10] This hyperlipidemia was associated with increased sperm cholesterol content, reduced acrosomal reaction, and impaired motility despite normal sperm morphology.^[78] The acrosomal membrane revealed a significant increase in filipin-cholesterol complexes, and disruption of the blood-testis barrier was observed.^[79] Interestingly, supplementing the diet with 7% olive oil reversed these effects, likely due to the antioxidant properties of olive oil that prevented lipotoxicity.^[80]

In rodent models, transgenic mice were employed to study the effect of hyperlipidemia on male fertility. Liver X receptors (LXRs) are nuclear receptors with transcription factor function that play an important role in lipid homeostasis and male fertility. The LXR-α isoform is expressed in Leydig and germ cells, while the LXR-β isoform is found in Sertoli cells, both of which are stimulated by oxysterols during oxidative stress to regulate lipid homeostasis.^[81] Transgenic mice lacking both isoforms of LXR developed fertility issues as they aged, as defective cholesterol metabolism resulted in CE accumulation in Sertoli cells, leading to testicular dysfunction in later life. Using this transgenic mouse, a diet-induced post-testicular infertility model was developed. When fed a cholesterol-rich diet, these knockout mice developed severe sperm abnormalities, similar to hypercholesterolemic rabbits, including abnormal morphology, decreased motility, and capacitation failure.^[81] Similarly, rats on a high cholesterol diet for 120 days showed a significant increase in plasma LDL, reduced sperm function, smaller LCs, lower germ cell numbers, and decreased testosterone.^[10]

High intake of saturated fat in humans correlates with reduced sperm quality, directly proportional to the quantity consumed.^[4] In addition, increased LDL and total cholesterol cause endothelial dysfunction and vascular obstruction, which can lead to erectile dysfunction as elevated levels of inflammatory cytokines cause testicular endothelial dysfunction, impairing endothelium-dependent relaxation of vascular beds.^[82,83] Sperm oxidative stress damage showed a positive association with increased levels of cytochrome C and caspases 9 and 3, which points to heightened sperm apoptosis in infertile men.^[84]

ART studies reveal that oxidative stress-induced sperm damage contributes to 30–80% of male infertility cases. Therefore, sperm DNA fragmentation from high ROS is used as an infertility diagnostic marker.^[6] Additional indicators of testicular dysfunction include elevated testicular malondialdehyde (MDA), a byproduct of lipid peroxidation, and reduced levels of key testicular antioxidants – glutathione (GSH) and superoxide dismutase (SOD).^[6] ART studies reveal that infertile men with high levels of ROS have a fivefold lower likelihood of achieving pregnancy than those with lower ROS levels.^[85] Obesity, frequently resulting from lipid imbalance and oxidative stress, contributes to systemic inflammation that worsens sperm DNA fragmentation due to altered redox homeostasis, further impairing male fertility.^[6]