The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding mammalian oocytes. Recently, numerous mutations in genes encoding ZP proteins have been shown to be possibly related to oocyte abnormality and female infertility; few reports have confirmed the functions of these mutations in living animal models. Here, we identified a novel heterozygous missense mutation (NM_001376231.1:c.1616C>T, p.Thr539Met) in ZP2 from a primary infertile female. We showed that the mutation reduced ZP2 expression and impeded ZP2 secretion in cell lines. Furthermore, we constructed the mouse model with the mutation (Zp2T541M) using CRISPR-Cas9. Zp2WT/T541M female mice had normal fertility though generated oocytes with the thin ZP, whereas Zp2T541M female mice were completely infertile due to degeneration of oocytes without ZP. Additionally, ZP deletion impaired folliculogenesis and caused female infertility in Zp2T541M mice. Our study not only expands the spectrum of ZP2 mutation sites but also, more importantly, increases the understanding of pathogenic mechanisms of ZP2 mutations.

The process of follicular development involves communications between oocyte and surrounding granulosa cells. FURIN is a member of the family of proprotein convertases that is involved in the activation of a large number of zymogens and proproteins by cleavage at its recognition motif. To investigate the functions of FURIN in female fertility, furinflox/flox (furfl/fl) mice were crossed with Zp3-Cre mice and Gdf9-Cre, respectively, to achieve oocyte-specific disruption of FURIN. Here we report for the first time that FURIN is dispensable for primordial follicle maintenance and activation but important for early secondary follicular development, as ablation of FURIN in oocytes caused failure of follicle development beyond the type 4 and/or 5a follicles in mutant mice, resulting in increased number of early secondary follicles and the severely decreased number of mature follicles, thus anovulation and infertility. We also found that the developmental arrest of early secondary follicles might be rooted in the loss of the mature form of ADAMTS1 (85-kDa prodomain truncated) and compromised proliferation of granulosa cells in mutant mice. Taken together, our data highlight the importance of FURIN in follicle development beyond the early secondary follicle stage and indicate that compromised FURIN function leads to follicular dysplasia and female infertility in mice.

Acephalic spermatozoa syndrome is a severe teratozoospermia that leads to male infertility. Our previous work showed that biallelic SUN5 mutations are responsible for acephalic spermatozoa syndrome in about half of affected individuals, while pathogenic mechanisms in the other individuals remain to be elucidated. Here, we identified a homozygous nonsense mutation in the testis-specific gene PMFBP1 using whole-exome sequencing in a consanguineous family with two infertile brothers with acephalic spermatozoa syndrome. Sanger sequencing of PMFBP1 in ten additional infertile men with acephalic spermatozoa syndrome and without SUN5 mutations revealed two homozygous variants and one compound heterozygous variant. The disruption of Pmfbp1 in male mice led to infertility due to the production of acephalic spermatozoa and the disruption of PMFBP1's cooperation with SUN5 and SPATA6, which plays a role in connecting sperm head to the tail. PMFBP1 mutation-associated male infertility could be successfully overcome by intracytoplasmic sperm injection (ICSI) in both mouse and human. Thus, mutations in PMFBP1 are an important cause of infertility in men with acephalic spermatozoa syndrome.

Female infertility is a worldwide concern and the etiology of infertility has not been thoroughly demonstrated. Although the mouse is a good model system to perform functional studies, the differences between mouse and human also need to be considered. The objective of this study is to elucidate the different molecular mechanisms underlying oocyte maturation and fertilization between human and mouse.

Premature ovarian failure (POF), a major cause of female infertility, is a complex disorder, but the molecular mechanisms underlying the disorder are only poorly understood. Here we report that protein kinase CK2 contributes to maintaining follicular survival through PI3K/AKT pathway and DNA damage response pathway. Targeted deletion of CK2β in mouse oocytes from the primordial follicle stage resulted in female infertility, which was attributed to POF incurring by massive follicle atresia. Downregulated PI3K/AKT signaling was found after CK2β deletion, indicated by reduced level of phosphorylated AKT (S473, T308, and S129) and altered AKT targets related to cell survival. Further studies discovered that CK2β-deficient oocytes showed enhanced γH2AX signals, indicative of accumulative unrepaired DSBs, which activated CHK2-dependant p53 and p63 signaling. The suppressed PI3K/AKT signaling and failed DNA damage response signaling probably contribute to large-scale oocyte loss and eventually POF. Our findings provide important new clues for elucidating the mechanisms underlying follicle atresia and POF.

The global delay in women's reproductive age has raised concerns about age-related infertility. The decline in oocyte quality is a limiting factor of female fertility, yet there are currently no strategies to preserve oocyte quality in aged women. Here, we investigated the effects of growth hormone (GH) supplementation on aneuploidy of aged oocytes.

In humans, non-obstructive azoospermia (NOA) is a major cause of male infertility. However, the aetiology of NOA is largely unknown. Previous studies reported that protein CK2β was abundantly and broadly expressed in spermatogenic cells. Here, we investigate whether protein CK2β participates in spermatogenesis.

Tuberous sclerosis complex 1 (Tsc1) is a tumor suppressor negatively regulating mammalian target of rapamycin complex 1 (mTORC1). It is reported that mice lacking Tsc1 gene in oocytes show depletion of primordial follicles, resulting in premature ovarian failure and subsequent infertility. A recent study indicated that deletion of Tsc1 in somatic cells of the reproductive tract caused infertility of female mice. However, it is not known whether specific disruption of Tsc1 in granulosa cells influences the reproductive activity of female mice. To clarify this problem, we mated Tsc1(flox/flox) mice with transgenic mice strain expressing cyp19-cre which exclusively expresses in granulosa cells of the ovary. Our results demonstrated that Tsc1(flox/flox); cyp19-cre mutant mice were fertile, ovulating more oocytes and giving birth to more pups than control Tsc1(flox/flox) mice. Progressive accumulation of corpora lutea occurred in the Tsc1(flox/flox); cyp19-cre mutant mice with advanced age. These phenotypes could be explained by the elevated activity of mTORC1, as indicated by increased phosphorylation of rpS6, a substrate of S6 in the Tsc1(flox/flox); cyp19-cre mutant granulosa cells. In addition, rapamycin, a specific mTORC1 inhibitor, effectively rescued the phenotype caused by increased mTORC1 activity in the Tsc1(cko) ovaries. Our data suggest that conditional knockout of Tsc1 in granulosa cells promotes reproductive activity in mice.

SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5-/- mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility.

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.

Oocyte activation inefficiency is one of the reasons for female infertility and Ca2+ functions play a critical role in the regulation of oocyte activation. We used various inhibitors of Ca2+ channels located on the membrane, including sarcoplasmic/ endoplasmic reticulum Ca2+ATPases (SERCAs, the main Ca2+ pumps which decrease the intracellular Ca2+ level by refilling Ca2+ into the sarcoplasmic reticulum), transient receptor potential (TRP) ion channel subfamily member 7 (TRPM7, a Ca2+/Mg2+-permeable non-selective cation channel), T-type Ca2+ channels and calcium channel Orai1, to investigate their roles in [Ca2+]i oscillation patterns and mitochondrial membrane potential during oocyte activation by real-time recording. Our results showed that SERCAs, TRPM7 and T-type Ca2+ channels were important for initiation and maintenance of [Ca2+]i oscillations, which was required for mitochondrial membrane potential elevation during oocyte activation, as well as oocyte cytoskeleton stability and subsequent embryo development. Increasing the knowledge of calcium transport may provide a theoretical basis for improving oocyte activation in human assisted reproduction clinics.

Liver Kinase b1 (LKB1/STK11)is a tumor suppressor responsible for the Peutz-Jeghers syndrome, an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. Besides, the C allele of a SNP in the Lkb1 gene impedes the likelihood of ovulation in polycystic ovary syndrome (PCOS) in women treated with metformin, a known LKB1-AMPK activator. It is very likely that LKB1 plays roles in female fertility. To identify the physiological functions of LKB1 in the mouse ovary, we selectively disrupted LKB1 in oocytes by the Cre-LoxP conditional knockout system and found that Lkb1fl/fl; Gdf9-Cre mice were severely subfertile with significantly enlarged ovaries compared to Lkb1fl/fl mice. Interestingly, without Lkb1 expression in oocytes from the primordial follicle stage, the entire primordial follicle pool was activated but failed to mature and ovulate, subsequently causing premature ovarian failure (POF). Further investigation demonstrated that elevated mTOR signaling regulated by an AKT-independent LKB1-AMPK pathway was responsible for the excessive follicle activation and growth. Our findings reveal the role of LKB1 as an indispensable gatekeeper for the primordial follicle pool, offer new functional understanding for the tumor suppressor genes in reproductive organs, and might also provide valuable information for understanding POF and infertility.