Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

Men and women become infertile with age, but the mechanism of declining male fertility, more specifically, the decrease in in sperm quality, is not well known. Citrate synthase (CS) is a core enzyme of the mitochondrial tricarboxylic acid (TCA) cycle, which directly controls cellular function. Extra-mitochondrial CS (eCS) is produced and abundant in the sperm head; however, its role in male fertility is unknown. We investigated the role of eCS in male fertility by producing eCs-deficient (eCs-KO) mice. The initiation of the first spike of Ca2+ oscillation was substantially delayed in egg fused with eCs-KO sperm, despite normal expression of sperm factor phospholipase C zeta 1. The eCs-KO male mice were initially fertile, but the fertility dropped with age. Metabolomic analysis of aged sperm revealed that the loss of eCS enhances TCA cycle in the mitochondria with age, presumably leading to depletion of extra-mitochondrial citrate. The data suggest that eCS suppresses age-dependent male infertility, providing insights into the decline of male fertility with age.

Correction: Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Suppression of Non-Random Fertilization by MHC Class I Antigens.

Hermaphroditic invertebrates and plants have a self-recognition system on the cell surface of sperm and eggs, which prevents their self-fusion and enhances non-self-fusion, thereby contributing to genetic variation. However, the system of sperm-egg recognition in mammals is under debate. To address this issue, we explored the role of major histocompatibility complex class I (MHC class I, also known as histocompatibility 2-Kb or H2-Kb and H2-Db in mice) antigens by analyzing H2-Kb-/-H2-Db-/-ß2-microglobulin (ß2M)-/- triple-knockout (T-KO) male mice with full fertility. T-KO sperm exhibited an increased sperm number in the perivitelline space of wild-type (WT) eggs in vitro. Moreover, T-KO sperm showed multiple fusion with zona pellucida (ZP)-free WT eggs, implying that the ability of polyspermy block for sperm from T-KO males was weakened in WT eggs. When T-KO male mice were intercrossed with WT female mice, the percentage of females in progeny increased. We speculate that WT eggs prefer fusion with T-KO sperm, more specifically X-chromosome-bearing sperm (X sperm), suggesting the presence of preferential (non-random) fertilization in mammals, including humans.

Membrane protein CD9 is repositioned and released to enhance uterine function.

Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.

Seminal vesicle protein SVS2 is required for sperm survival in the uterus.

In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2(-/-) male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.

Suppressive Role of Lactoferrin in Overweight-Related Female Fertility Problems.

The secretory glycoprotein lactoferrin (LF) is suggested to ameliorate overweight regardless of non-genetic or genetic mechanisms. Although maternal overweight represents a key predictor of offspring growth, the efficacy of LF on fertility problems in overweight and obese mothers remains unknown. To address this issue, we examined the effect of LF ingestion by analyzing overweight mice (Institute of Cancer Research (ICR) mice with high-fat diets; HF mice) and obese mice (leptin-deficient mice with type II diabetes; ob/ob mice). Plasma insulin, leptin, glucose, and cholesterol levels were measured, and thermal imaging and histological analysis were employed. The litter size of HF females was reduced due to miscarriage, which was reversed by LF ingestion. In addition, LF ingestion suppressed overweight prevalence in their offspring. The component analysis of the maternal blood demonstrated that glucose concentration in both HF females and their offspring was normalized by LF ingestion, which further standardized the concentration of insulin, but not leptin. LF ingestion was unable to reverse female infertility in ob/ob mice, although their obesity and uterine function were partially improved. Our results indicate that LF upregulates female fertility by reinforcing ovarian and uterine functions in females that are overweight due to caloric surplus.

Innate immune system still works at diapause, a physiological state of dormancy in insects.

Diapause is most often observed in insects and is a physiologically dormant state different from other types of dormancy, such as hibernation. It allows insects to survive in harsh environments or extend longevity. In general, larval, pupal, or adult non-diapausing insects possess an innate immune system preventing the invasion of microorganisms into their bodies; however, it is unclear whether this system works under the dormant condition of diapause. We here report the occurrence of innate cellular reactions during diapause using pupae of a giant silkmoth, Samia cynthia pryeri. Scanning electron microscopic analysis demonstrated the presence of two major types of cells in the body fluid isolated from the thoracic region of a pupa. Phagocytosis and encapsulation, characteristics of innate cellular reactions, by these cells were observed when latex beads as foreign targets were microinjected into the internal portion of a pupa. Such behavior by these cells was still observed even when pupae were continuously chilled at 4°C. Our results indicate that innate cellular reactions can work in diapausing insects in a dormant state.

Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition.

In mammals, uterine epithelium is remodeled cyclically throughout adult life for pregnancy. Despite the expression of CD9 in the uterine epithelium, its role in maternal reproduction is unclear. Here, we addressed this issue by examining uterine secretions collected from patients undergoing fertility treatment and fertilization-competent Cd9(-/-) mice expressing CD9-GFP in their eggs (Cd9(-/-)TG). CD9 in uterine secretions was observed as extracellular matrix-like feature, and its amount of the secretions associated with repeated pregnancy failures. We also found that the litter size of Cd9(-/-)TG female mice was significantly reduced after their first birth. Severely delayed re-epithelialization of the endometrium was then occurred. Concomitantly, vascular endothelial growth factor (VEGF) was remarkably reduced in the uterine secretions of Cd9(-/-)TG female mice. These results provide the first evidence that CD9-mediated VEGF secretion plays a role in re-epithelialization of the uterus.

CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte.

When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

ß-catenin is a molecular switch that regulates transition of cell-cell adhesion to fusion.

When a sperm and an oocyte unite upon fertilization, their cell membranes adhere and fuse, but little is known about the factors regulating sperm-oocyte adhesion. Here we explored the role of ß-catenin in sperm-oocyte adhesion. Biochemical analysis revealed that E-cadherin and ß-catenin formed a complex in oocytes and also in sperm. Sperm-oocyte adhesion was impaired when ß-catenin-deficient oocytes were inseminated with sperm. Furthermore, expression of ß-catenin decreased from the sperm head and the site of an oocyte to which a sperm adheres after completion of sperm-oocyte adhesion. UBE1-41, an inhibitor of ubiquitin-activating enzyme 1, inhibited the degradation of ß-catenin, and reduced the fusing ability of wild-type (but not ß-catenin-deficient) oocytes. These results indicate that ß-catenin is not only involved in membrane adhesion, but also in the transition to membrane fusion upon fertilization.