Ovarian differentiation relies on the accurate and orderly expression of numerous related genes. Forkhead box protein L2 (FOXL2) is one of the earliest ovarian differentiation markers and transcription factors. In sex determination, FOXL2 maintains the differentiation of the female pathway by inhibiting male differentiation genes, including SOX9 and SF1. In addition, FOXL2 promotes the synthesis of follicle-stimulating hormone and anti-Müllerian hormone to support follicle development. Mutations in FOXL2 are associated with numerous female reproductive diseases. A comprehensive and in-depth study of FOXL2 provides novel strategies for the diagnosis and treatment of such diseases. This review discusses the mechanism of FOXL2 in female sex differentiation and maintenance, hormone synthesis, and disease occurrence and reveals the role of FOXL2 as a central factor in female sex development and fertility maintenance. This review will serve as a reference for identifying novel targets of other regulatory factors interacting with FOXL2 in female sex determination and follicle development and for the diagnosis and treatment of female reproductive diseases.

Summary Sentence

This review explores the mechanism of FOXL2 in female sex differentiation and maintenance, hormone synthesis, and disease occurrence and depicts the role of FOXL2 as a central factor in female sex development and fertility maintenance.

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Unlike differentiated somatic cells, which possess elongated mitochondria, undifferentiated cells, such as those of preimplantation embryos, possess round, immature mitochondria. Mitochondrial morphology changes dynamically during cell differentiation in a process called mitochondrial maturation. The significance of the alignment between cell differentiation and mitochondrial maturity in preimplantation development remains unclear. In this study, we analyzed mouse embryos into which liver-derived somatic mitochondria were introduced (SM-embryos). Most SM-embryos were arrested at the two-cell stage. Some of the introduced somatic mitochondria became round, while others remained elongated and large. RNA-sequencing revealed a disruption of both minor and major zygotic gene activation (ZGA) in SM-embryos. Minor ZGA did not terminate before major ZGA, and the onset of major ZGA was inhibited, as shown by histone modification analyses of histone H3 lysine 4 trimethylation and histone H3 lysine 27 acetylation. Further analysis of metabolites involved in histone modification regulation in SM-embryos showed a significantly lower NAD⁺/NADH ratio in SM-embryos than in control embryos. Additionally, the mitochondrial membrane potential, an indicator of mitochondrial function, was lower in SM-embryos than in control embryos. Our results demonstrated that introducing somatic mitochondria into an embryo induces mitochondrial dysfunction, thereby disrupting metabolite production, leading to a disruption in ZGA and inducing developmental arrest. Our findings reveal that the alignment between cell differentiation and mitochondrial maturity is essential for early embryonic development.

Summary Sentence

Mismatch between cell differentiation and mitochondrial maturity prevents transition from minor to major ZGA in mouse embryos.

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In vitro fertilization is a widely used assisted reproductive technology to achieve a successful pregnancy. However, the acquisition of oxidative stress in embryo in vitro culture impairs its competence. Here, we demonstrated that a nuclear coding gene, methyltransferase-like protein 7A, improves the developmental potential of bovine embryos. We found that exogenous methyltransferase-like protein 7A modulates expression of genes involved in embryonic cell mitochondrial pathways and promotes trophectoderm development. Surprisingly, we discovered that methyltransferase-like protein 7A alleviates mitochondrial stress and DNA damage and promotes cell cycle progression during embryo cleavage. In summary, we have identified a novel mitochondria stress eliminating mechanism regulated by methyltransferase-like protein 7A that occurs during the acquisition of oxidative stress in embryo in vitro culture. This discovery lays the groundwork for the development of methyltransferase-like protein 7A as a promising therapeutic target for in vitro fertilization embryo competence.

Summary Sentence

We describe a molecule acts in the pre-implantation period to attenuate oxidative stress that enhances embryo development to the blastocyst stage and subsequent pregnancy in cattle.

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Endometriosis is a common gynecological disorder, whose pathogenesis remains incompletely understood. Macrophages, a key type of immune cell, are pivotal in the context of endometriosis. This study seeks to explore the interactions between endometriotic cells and macrophages. Quantitative real-time PCR (qRT-PCR) and Western blot experiments were employed to detect phosphatase and tensin homolog (PTEN) expression. Glucose consumption, lactate production, extracellular acidification rate, and oxygen consumption rate levels were used to assess cellular glycolytic capacity. The interaction between conditioned media from ectopic endometrial stromal cells (EESCs) and macrophages was investigated through co-culture experiments. The expression of M2 macrophage marker proteins and inflammatory factors was detected via qRT-PCR, immunofluorescence staining, and enzyme-linked immunosorbent assay. Cellular functions were evaluated using Cell Counting Kit-8, 5-Ethynyl-2′-deoxyuridine (EdU), and wound healing assays. We found that PTEN deficiency promoted the glycolytic activity of EESCs. Simultaneously, it significantly promoted the macrophages' polarization toward the M2 phenotype, demonstrated by increased expression of M2 markers (differentiation 206 (CD206), CD163, and (C-C motif) ligand 22 (CCL22)). Further studies revealed that PTEN-deficient EESCs increased the level of CCL2 via promoting glycolytic activity, which was reversed by glycolytic inhibitor. Moreover, lactate and conditioned media from overexpressed CCL2 EESCs facilitated M2 polarization of macrophages, while 2-deoxy-D-glucose reversed the promoting effect. Furthermore, lactate-facilitated macrophages promoted the proliferation and migration abilities of EESCs. PTEN deficiency induces M2 macrophage polarization by promoting glycolytic activity in EESCs, which deepens the knowledge of the pathophysiology of endometriosis and provides novel insights into its treatment.

Summary Sentence

PTEN deficiency induces polarization of M2 macrophages by promoting glycolytic activity in EESCs, which is crucial for deepening the understanding of the pathophysiology of endometriosis and providing new insights for the treatment of endometriosis.

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The physiological and clinical importance of motile cilia in reproduction is well recognized; however, the specific role they play in transport through the oviduct and how ciliopathies lead to subfertility and infertility are still unclear. The contribution of cilia beating, fluid flow, and smooth muscle contraction to overall progressive transport within the oviduct remains under debate. Therefore, we investigated the role of cilia in the oviduct transport of preimplantation eggs/embryos using a combination of genetic and advanced imaging approaches. We show that the region of the oviduct where cumulus–oocyte complex circling occurs, around the time of fertilization, is correlated with asymmetrical mucosal fold arrangement and non-radially distributed ciliated epithelium. Our results suggest that motile cilia, as well as mucosal fold asymmetry, may contribute to the local flow fields that help steer luminal contents away from the epithelial walls. We also present, in vivo, volumetric evidence of delayed egg transport in a genetic mouse model with disrupted motile cilia function in the female reproductive system. Females with Dnah5 deleted in the oviduct epithelium are subfertile and demonstrate disrupted motile cilia activity within the oviduct mucosa. Fifty percent of Dnah5 mutant females have delayed egg transport where cumulus–oocyte complexes did not progress to the ampulla at the expected time point and remained within the ovarian bursa. The integration of advanced imaging with genetic dysfunction of motile cilia provides valuable insights into oviductal transport. Potentially, these data could be valuable for better understanding and management of tubal pathologies and human infertility.

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Summary Sentence

Disruption of motile cilia function in the oviduct affects egg/embryo transport and fertility in mice revealed by genetic and advanced imaging approaches.

Periodic increases in cytosolic calcium concentration (Ca²⁺ oscillations) during mammalian fertilization induce all the events collectively known as egg activation. The sperm-specific phospholipase C, Phospholipase C zeta 1 (PLCZ1) represents the “sperm factor” vital for initiating the persistent Ca²⁺ oscillations in mammals. Despite sequence conservation, the Ca²⁺ oscillation-inducing properties of the enzyme differ vastly among species, and this is particularly salient between mouse and rat PLCZ1, where the activities vary at least one order of magnitude in favor of the former. As previously shown, injecting wild-type rat Plcz1 mRNA into metaphase II mouse eggs induced delayed Ca²⁺ oscillations with low specific activity compared to the homologous mouse Plcz1 mRNA. We, therefore, sought to uncover the factor(s) diversifying these enzymes by swapping functional domains between species, creating chimeric PLCZ1s. When injected into mouse metaphase II eggs, mouse Plcz1 mRNA with the whole- or part of the EF-hand domains swapped with the rat showed a substantial reduction in activity compared to WT. Consistently, the opposite exchange enhanced the rat's enzyme activity. EF-hand domains 1 and 2 seemed to underlie most differences, and mutations of the divergent amino acids within these domains, substitutions for Glu^{(m-30; r-29)} and Gln^{(m-58; r-57)}, changed the activity of both species' PLCZ1s in opposite directions. Collectively, our findings support the view that differences in the sequences of EF-hand domains, especially in several of its charged residues, underpin the distinct PLCZ1 activities between these species, revealing the gametes and species' adaptability to optimize the fertilization signal and early development.

Summary Sentence

Differences in the amino acid composition of the EF-hand domain of mouse and rat PLCZ1s, especially in the charged residues, determine the activities of these enzymes that translate into the distinct Ca²⁺ oscillation patterns of fertilization.

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Granulosa and thecal layer cells play important roles in the post-hatching follicular growth in laying birds. To examine the biochemical processes of granulosa and thecal layers associated with follicular growth, the technique of data independent acquisition was used in this study to explore protein profiling in granulosa and thecal layers from growing follicles in laying ducks. We identified and quantitatively analyzed 8032 proteins in granulosa cells and 9552 proteins in thecal layer cells. Hierarchical clustering of the resulting profiles revealed differential changes of expression of proteins linked to cell metabolism, signaling, cell junction, especially in steroid synthesis, peroxisome proliferator-activated receptor, and gap junction signaling pathway at different stages of follicles. The highest expression of proteins related to gap junction and peroxisome proliferator-activated receptor signaling pathway occurred in granulosa cells of 3–6 mm or 6–8 mm follicles. In granulosa cells, decreases in the enzymes that catalyze the transformation of estrone into estradiol and proteins related to calcium transport and apoptosis occurred during follicular growth. As follicles grew, proteins related to androgens biosynthesis and involved in gap junction and peroxisome proliferator-activated receptor signaling pathway decreased in the thecal layer cells. Three main group functional clusters extracted from the protein–protein interaction network, were mainly responsible for apoptosis, steroid hormone biosynthesis, and the peroxisome proliferator-activated receptor signaling pathway. These proteomic data provide a holistic framework for understanding how diverse biochemical processes in granulosa cells and thecal layer cells are coordinated at the cellular level during follicular growth in laying birds.

Summary Statement

The identified factors and bidirectional communication of local signals originating from the granulosa and thecal layer cells were obtained in different stages of duck follicles by data-independent acquisition.

Graphical Abstract

Diet-induced obesity can cause long-term alterations in ovarian functions, but the acute effects of obesogenic diets on the follicular cells and their progression over time, when intake is continued and obesity develops, remain unclear. We aimed to determine the onset and progression of changes in the granulosa cell transcriptomic profile after starting a high-fat/high sugar (HFHS)-diet feeding in mice. We also examined the changes in oocyte lipid droplet content and mitochondrial ultrastructural abnormalities. Swiss (outbred) mice were sacrificed at 24 h, 3 days, and at 1, 4, 8, 12, and 16 weeks of feeding HFHS and control diets. Lipid droplet content significantly increased in the HFHS oocytes within 24 h compared to controls (P < 0.05). Oocyte mitochondrial abnormalities only increased starting from 8 weeks. Granulosa RNA-seq revealed altered transcriptomic gene-set enrichments (GO terms and KEGG pathways, P_adj < 0.05) already at 3 days and 1 week indicating acute endoplasmic reticulum unfolded protein responses, with concomitant fluctuations in several cellular metabolic pathways and gene sets related to mitochondrial bioenergetic functions, some of which persisted after 8 weeks. Interestingly, the short- and long-term patterns of changes in cytochrome P450, steroid hormone biosynthesis, retinol metabolism, bile acid metabolism, fatty acid metabolism, and Pi3K/Akt signaling pathways were most prominent and highly correlated; all being acutely upregulated, then chronically downregulated. These results show that the impact of obesogenic diet on the oocyte and granulosa cells is prompt, while the response depends on the duration of feeding and occurs in a multiphasic cascade together with a progressive deterioration in oocyte quality.

Summary Sentence

Granulosa cells undergo acute stress responses within a few days on an obesogenic diet, inhibiting retinol metabolism, and bile acid, fatty acid, and steroid hormone synthesis pathways after long-term exposure, with late oocyte mitochondrial defects.

Graphical Abstract

Reproductive success requires accurately timed remodeling of the cervix to orchestrate the maintenance of pregnancy, the process of labor, and birth. Prior work in mice established that a combination of continuous turnover of fibrillar collagen and reduced formation of collagen cross-links allows for the gradual increase in tissue compliance and delivery of the fetus during labor. However, the mechanism for continuous collagen degradation to ensure turnover during cervical remodeling is still unknown. This study demonstrates the functional role of extracellular and intracellular collagen degradative pathways in two different settings of cervical remodeling: physiological term remodeling and inflammation-mediated premature remodeling. Extracellular collagen degradation is achieved by the activity of fibroblast-derived matrix metalloproteases MMP14, MMP2, and fibroblast activation protein (FAP). In parallel, we demonstrate the function of an intracellular collagen degradative pathway in fibroblast cells mediated by the collagen endocytic mannose receptor type-2 (MRC2). These pathways appear to be functionally redundant as loss of MRC2 does not obstruct collagen turnover or cervical function in pregnancy. While both extracellular and intracellular pathways are also utilized in inflammation-mediated premature cervical remodeling, the extracellular collagen degradation pathway uniquely employs fibroblast and immune-cell-derived proteases. In sum, these findings identify the dual utilization of two distinct degradative pathways as a failsafe mechanism to achieve continuous collagen turnover in the cervix, thereby allowing dynamic shifts in cervical tissue mechanics and function.

Summary Sentence

Two distinct collagen degradation pathways allow for cervical collagen turnover in non-pregnancy, term pregnancy, and inflammation-mediated preterm birth.

Graphical Abstract

The creatine (Cr) biosynthesis pathway buffers adenosine triphosphate in metabolically active tissues. We investigated whether sex of fetus and day of gestation influence Cr in endometrial and conceptus tissues from gilts on days 60 and 90 (n = 6 gilts/day) of gestation. Uterine and conceptus tissues associated with one male and one female fetus from each gilt were analyzed for creatine, messenger RNAs (mRNAs), and proteins for Cr biosynthesis. Total Cr decreased in amniotic fluid but increased in allantoic fluid between days 60 and 90 of gestation for male (P < 0.05) but not for female fetuses (P > 0.05). Endometrial expression of creatine kinase, muscle (CKM), creatine kinase mitochondrial type 1 (CKMT1), and solute carrier family 6, member 8 (SLC6A8) mRNAs increased (P < 0.05) between days 60 and 90 only for female fetuses. On day 60, expression of creatine kinase, brain (CKB) and CKMT1 mRNAs was greater (P < 0.05) for placentae of female than male fetuses. Livers of male fetuses had greater expression of arginine:glycine amidinotransferase (AGAT) and CKB than for females on day 60, while kidneys of female fetuses had greater expression of guanidinoacetate-N-methyltransferase (GAMT) than male fetuses on day 90 (P < 0.05). Localization of GAMT, CKB, CKMT1, and SLC6A8 proteins to uterine and chorionic epithelium was not influenced by gestational age or fetal sex. Arginineglycine amidinotransferase localized to fetal kidneys and appeared greater on day 90 than on day 60 in both sexes. Thus, expression of the creatine–creatine kinase–phosphocreatine system at the uterine–conceptus interface is affected by gestational age and fetal sex to influence energy homeostasis in pigs.

Summary Sentence

Creatine is abundant in maternal and fetal plasma and fetal fluids, and enzymes required for metabolism of creatine are expressed in uteri, placentae, and fetuses of pigs indicating a role for creatine in fetal–placental development.

Graphical Abstract

The decidual endometrial stromal cells play a critical role in the establishment of uterine receptivity and pregnancy in human. Our previous studies demonstrate that protein tyrosine phosphatase 2 SHP2 is highly expressed in decidualized cells and governs the decidualization progress. However, the role and mechanism of SHP2 in the function of decidual cells remain unclear. Here, we screened proteins interacting with SHP2 in decidual hTERT-immortalized human endometrial stromal cells (T-HESCs) and identified Hypoxia-inducible factor-1 (HIF-1) signaling pathway as a potential SHP2-mediated signaling pathway through proximity-dependent biotinylation (BioID) analysis. Immunoprecipitation (Co-IP) revealed an interaction between SHP2 and HIF-1α, which colocalized to the nucleus in decidual cells. Furthermore, the SHP2 expression correlated with the transcriptional activation of HIF-1α and its downstream genes Beta-enolase (Eno3), Pyruvate kinase 2 (Pkm2), Aldolase C (Aldoc), and Facilitative glucose transporter 1 (Glut1). Knockdown or inhibition of SHP2 significantly reduced the mRNA and protein levels of HIF-1α and its downstream genes, as well as lactate production in decidual cells. We also established a hypoxia model of T-HESCs and 293 T cells and found that hypoxic treatment induced the expression of SHP2 and HIF-1α, which colocalized in the nucleus. SHP2 forced-expression rescued the inhibitory effects of SHP2 deficiency on HIF-1α expression and lactate production. Finally, SHP2 binds to the promoter regions of HIF-1α and its target genes (Eno3, Pkm2, Aldoc, and Glut1). Collectively, our results suggest that SHP2 influences the function of decidual cells by HIF-1α signaling and provide a novel function mechanism of decidual stromal cells.

Summary Sentence

SHP2 mediates HIF-1 signaling and lactate metabolism within decidual cells by regulating the transcriptional activation of HIF-1α and its downstream genes.

Graphical Abstract

Relationships between anti-Müllerian hormone concentrations and subsequent ovarian stimulation outcomes have been demonstrated in several mammalian species, but comprehensive reports are lacking in felids. Our objective was to characterize relationships between anti-Müllerian hormone concentrations and responses to exogenous gonadotropin stimulation in cheetahs and domestic cats. Blood samples collected before stimulation were used to measure serum anti-Müllerian hormone concentrations, which were compared to post-stimulation outcomes, including counts of retrievable oocytes or ovulation sites, oocyte quality, embryonic cleavage after in vitro fertilization, and progestogen concentrations. Anti-Müllerian hormone concentrations were also compared to outcomes in domestic cats induced to ovulate by mechanical stimulation of the vagina and cervix (simulated coitus). Greater anti-Müllerian hormone concentrations were associated with greater ovulatory response, progestogen production, and embryonic cleavage success among gonadotropin-treated cheetahs, and with greater ovulatory response among gonadotropin-treated domestic cats. Associations were moderated by age, with anti-Müllerian hormone concentration generally a greater determinant of these outcomes in older animals. Anti-Müllerian hormone concentrations alone could distinguish domestic cats with high and low ovulatory responses to exogenous hormones. However, this marker was unrelated to ovulatory response in domestic cats after simulated coitus. These results demonstrate the potential for anti-Müllerian hormone concentrations to predict responses of cheetahs and domestic cats to ovarian stimulation treatment commonly used in assisted reproductive technologies. Associations between anti-Müllerian hormone concentrations and ovarian stimulation outcomes in these species might reflect relationships between anti-Müllerian hormone concentration and antral follicle count or oocyte/embryo cellular function, as reported in other mammals; however, this remains to be tested.

Summary Sentence

Serum AMH concentrations are associated with subsequent ovarian stimulation outcomes in cheetahs and domestic cats, including progestogen concentrations and in vitro embryonic cleavage in cheetahs and counts of ovulations in both cheetahs and cats.

Graphical Abstract

Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of noncoding mutations remains largely unexplored. To evaluate the effects of noncoding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8. We then used CRISPR-Cas9 to generate a mouse line where 26 kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting mild increase in the DNA–DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, a higher frequency of histological abnormalities, a drop of 47–52% in efficiency of meiosis, a 15–18% reduction in efficiency of spermatogenesis, and, consistently, a 12–28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that noncoding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a topologically associated domain boundary can have a negative impact on sperm production and highlight the importance of considering noncoding mutations in the analysis of patients with male infertility.

Summary Sentence

Deletion of an evolutionarily conserved TAD boundary near spermatogenesis-related genes in mice alters local gene expression and affects sperm production.

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