Follicular development is a critical process in reproductive biology that determines the number of oocytes and interacts with various cells within the follicle (such as oocytes, granulosa cells, cumulus cells, and theca cells), and plays a vital role in fertility and reproductive health because of the dogma of a limited number of oogonia. Dysregulation of follicular development can lead to infertility problems and other reproductive disorders. To explore the physiological and pathological mechanisms of follicular development, immunology-based methods, microarrays, and next-generation sequencing have traditionally been used for characterization at the tissue level. However, with the proliferation of single-cell techniques, research has uncovered unique molecular mechanisms in individual cells that have been masked by previous holistic analyses. In this review, we briefly summarize the achievements and limitations of traditional methods in the study of follicular development. Simultaneously, we focus on how to understand the physiological process of follicular development at the single-cell level and reveal the pathological mechanisms and relevant intervention targets. Moreover, we also delineate the limitations and application prospects of single-cell sequencing in follicular development research.

Summary Sentence

Single cell sequencing research on ovarian aging and ovarian dysfunction.

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Gene knockout experiments have shown that many genes are dispensable for a given biological function. In this review, we make an assessment of male and female germ cell-specific genes dispensable for the function of reproduction in mice, the inactivation of which does not affect fertility. In particular, we describe the deletion of a 1 Mb block containing nineteen paralogous genes of the oogenesin/Pramel family specifically expressed in female and/or male germ cells, which has no consequences in both sexes. We discuss this notion of dispensability and the experiments that need to be carried out to definitively conclude that a gene is dispensable for a function.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26 days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.

Summary Sentence

Bacterial-induced endometritis impairs conceptus development by altering histotroph composition and conceptus gene expression in dairy cattle.

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In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus–oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or superGDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

Summary Sentence

Culture-inflicted disturbances on cumulus–oocyte complex glucose metabolism and mitochondrial respiration can be normalized when culturing under low oxygen (5%) tension during pre-IVM.

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Multiple morphological abnormalities of the flagella, a severe form of asthenozoospermia, can lead to male infertility. Recent studies have implicated an association between human CFAP70 deficiency and multiple morphological abnormalities of the flagella; however, the underlying biological mechanism and supporting experimental evidence in animal models remain unclear. To address this gap, we used CRISPR/Cas9 technology to generate Cfap70-deficient mice to investigate the relationship between Cfap70 deficiency and multiple morphological abnormalities of the flagella. Our findings show that the loss of CFAP70 leads to multiple morphological abnormalities of the flagella and spermiogenesis defects. Specifically, the lack of CFAP70 impairs sperm flagellum biogenesis and head shaping during spermiogenesis. Late-step spermatids from Cfap70-deficient mouse testis exhibited club-shaped sperm heads and abnormal disassembly of the manchette. Furthermore, we found that CFAP70 interacts with DNAI1 and DNAI2; Cfap70 deficiency also reduces the level of AKAP3 in sperm flagella, indicating that CFAP70 may participate in the flagellum assembly and transport of flagellar components. These findings provide compelling evidence implicating Cfap70 as a causative gene of multiple morphological abnormalities of the flagella and highlight the consequences of CFAP70 loss on flagellum biogenesis.

Summary Sentence

We demonstrate Cfap70 as a causative gene of multiple morphological abnormalities of the flagella in mice, and the loss of CFAP70 results in abnormal sperm flagellum biogenesis by affecting the assembly and transport of flagellar components.

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The male external genitalia of the black rockfish (Sebastes schlegelii Hilgendorf, 1880) is a fleshy protrusion known as the urogenital papilla (UGP), which functions to deliver sperm into the female reproductive tract for internal fertilization. It is not known which genes regulate the development of the UGP. The aim of this study was to identify key genes that regulate the development of the UGP in black rockfish and to determine the distribution of androgen receptor gene (ar) in the UGP. A total of 26 adult males and 560 juvenile fish were used in the experiment, in which we divided all normally developing juveniles into normal development and androgen groups. We added methyltestosterone solution (100 µg/l) to the androgen group-treated fish tank, soaked for 2 h per day for 38 days, and sampled 5∼10 samples each time every 5 days during the culture process. Gene expression changes related to UGP were analyzed with tissue specificity between control and androgen groups during sex differentiation, adult male maturation, and the copulation stage (September to December) using real-time quantitative polymerase chain reaction. The expression of ar was also localized by two-color in situ hybridization in the UGP region of juvenile fish. Androgen treatment enhanced ar expression levels and the ar signal was stronger in the UGP region of both adult breeding fish and androgen-treated juvenile fish. This study provides insights into the regulation of the external genitalia of black rockfish and presents vital information for the artificial breeding of viviparous fish.

Summary Sentence

We found that the ara, arb, mafb and shha genes may play important roles in the development of the male reproductive system and mating organs in black rockfish.

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The mammalian epididymis is the organ for functional sperm maturation. In rodents, the initial segment, the most proximal region of the epididymis, plays a critical role in sperm maturation. The luminal epithelial differentiation and the following gene expression of the initial segment are regulated by the lumicrine signaling, a testis-epididymis transluminal secreted signaling. Adhesion G protein-coupled receptor G2 (ADGRG2) is expressed in the efferent duct and the initial segment epididymis. In the preceding study, Adgrg2 ablation decreased the expression of several genes expressed in the initial segment. Such downregulated genes include those known to be regulated by lumicrine signaling, suggesting the involvement of ADGRG2 in lumicrine signaling. The present study examined whether ADGRG2 is associated with the lumicrine signaling regulating epididymal initial segment differentiation and gene expression. Adgrg2-null mice were generated by CRISPR/CAS9-mediated genome editing. The postnatal differentiation of the Adgrg2-null male epididymal initial segment was histologically comparable with that of control wild-type animals. The RNA-seq of Adgrg2-null mice was performed together with those of efferent duct-ligated and W/Wv mice in both of which lumicrine signaling is defective. The comparative transcriptome analyses clarified that the expressions of genes expressed in the initial segment and regulated by lumicrine signaling were decreased by Adgrg2 nullification. However, the extent of such downregulations observed in Adgrg2null epididymis was not so prominent compared with those of lumicrine signaling deficient Nell2–/^–, efferent duct-ligated, or W/Wv mice. Collectively, these findings indicate that ADGRG2 is dispensable for the lumicrine regulation of epididymal initial segment differentiation.

Summary Sentence

The association of ADGRG2 with the lumicrine-mediated epididymal initial segment differentiation and gene expression was examined by generating Adgrg2-null mice and concluded to be dispensable.

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As in mammals, ovarian folliculogenesis in teleosts also consists of two phases: the primary growth (PG) and secondary growth (SG) phases, which are analogous to the preantral and antral phases respectively in mammals. In this study, we performed a proteomic analysis on zebrafish follicles undergoing the PG–SG transition aiming to identify factors involved in the event. Numerous proteins showed significant changes, and the most prominent one was Y-box binding protein 1 (YB-1; Ybx1/ybx1), a transcription factor and mRNA-binding protein. YB-1 belongs to the Y-box binding protein family, which also includes the gonad-specific YB-2. Interestingly, phylogenetic analysis showed no YB-2 homolog in zebrafish. Although ybx1 mRNA was expressed in various tissues, its protein Ybx1 was primarily produced in the gonads, similar to YB-2 in other species. In the ovary, Ybx1 protein started to appear in early follicles newly emerged from the germ cell cysts, reached the highest level in late PG oocytes, but decreased precipitously when the follicles entered the SG phase. In PG follicles, Ybx1 might function as a key component of the messenger ribonucleoprotein particles (mRNPs) in association with other RNA-binding proteins. Similar to mammalian YB-1, zebrafish Ybx1 also contains functional signals that determine its intracellular localization. In conclusion, Ybx1 may play dual roles of YB-1 and YB-2 in zebrafish. In the ovary, Ybx1 binds mRNAs to stabilize them while preventing their translation. At PG–SG transition, Ybx1 is removed to release the masked mRNAs for translation into functional proteins, leading to follicle activation.

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Insulin-like growth factor 1 (IGF1) is an essential regulator of mammalian follicle development and synergizes with follicle-stimulating hormone (FSH) to amplify its effects. In avian preovulatory follicles, IGF1 increases the expression of genes involved in steroidogenesis and progesterone and inhibin A production. The role of IGF1 in prehierarchal follicles has not been well studied in chickens. The aim of this study was to investigate the role of IGF1 in granulosa cells from prehierarchal follicles and to determine whether IGF1 and FSH synergize to promote follicle development. Granulosa cells of 3–5 and 6–8 mm prehierarchal follicles were cultured with IGF1 (0, 10, 100 ng/mL) in the presence or absence of FSH (0, 10 ng/mL). Cell proliferation, expression of genes important in follicle development (FSHR, IGF1R, AMH, STAR, CYP11A1, INHA, and INHBA), and progesterone production were evaluated following treatment. IGF1 treatment alone significantly increased STAR, CYP11A1, and INHBA mRNA expression and cell proliferation in granulosa cells of 6–8 mm follicles. IGF1 and FSH synergized to increase STAR mRNA expression in 6–8 mm follicles. IGF1 and FSH co-treatment were necessary to increase INHA mRNA expression in 6–8 mm follicles. Although IGF1 significantly increased the expression of genes involved in steroidogenesis, progesterone production in granulosa cells of 6–8 mm follicles was not affected. IGF1 did not affect AMH mRNA expression, although FSH significantly decreased AMH expression in granulosa cells of 3–5 mm follicles. These results suggest that IGF1 may act with FSH to promote follicle selection at the prehierarchal follicle stage.

Summary Sentence

Insulin-like growth factor 1 and follicle-stimulating hormone treatment of cultured granulosa cells from prehierarchal follicles promote expression of follicle development markers including STAR, CYP11A1, INHA, and INHBA.

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The mechanism underlying the initiation of parturition remains unclear. Cyclooxygenase 2 and prostaglandins in decidual membrane tissue play an important role in the “parturition cascade.” With the advancement of gestation, the expression of the transcriptional suppressor B lymphocyte-induced maturation protein 1 in the decidual membrane gradually decreases. Through chromatin immunoprecipitation sequencing, we found that B lymphocyte-induced maturation protein 1 has a binding site in the distal intergenic of PTGS2(COX2). Tripartite motif-containing protein 66 is a chromatin-binding protein that usually performs transcriptional regulatory functions by “reading” histone modification sites in chromatin. In this study, tripartite motif-containing protein 66 exhibits the same trend of expression as B lymphocyte-induced maturation protein 1 in the decidua during gestation. Moreover, the co-immunoprecipitation assay revealed that tripartite motif-containing protein 66 combined with B lymphocyte-induced maturation protein 1. This finding indicated that tripartite motif-containing protein 66 formed a transcription complex with B lymphocyte-induced maturation protein 1, which coregulated the expression of COX2. In animal experiments, we injected si-Blimp1 adenoviruses (si-Blimp1), Blimp1 overexpression plasmid (Blimp1-OE), and Trim66 overexpression plasmid (Trim66-OE) through the tail vein of mice. The results showed that B lymphocyte-induced maturation protein 1 and tripartite motif-containing protein 66 affected the initiation of parturition in mice. Therefore, the present evidence suggests that B lymphocyte-induced maturation protein 1 and tripartite motif-containing protein 66 partially participate in the initiation of labor, which may provide a new perspective for exploring the mechanism of term labor.

Summary Sentence

As pregnancy advanced, the expression of the transcription complex formed by BLIMP1 and TRIM66 decreased in the decidua, promoting the expression of COX2 and the production of PGF2alpha, thereby inducing the onset of parturition.

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The reproductive homeobox X-linked (Rhox) genes encode transcription factors that are expressed selectively in reproductive tissues including the testis, epididymis, ovary, and placenta. While many Rhox genes are expressed in germ cells in the mouse testis, only Rhox8 is expressed exclusively in the Sertoli cells during embryonic and postnatal development, suggesting a possible role of Rhox8 in embryonic gonad development. Previously, Sertoli cell–specific knockdown of RHOX8 resulted in male subfertility due to germ cell defects. However, this knockdown model was limited in examining the functions of Rhox8 as RHOX8 knockdown occurred only postnatally, and there was still residual RHOX8 in the testis. In this study, we generated new Rhox8 knockout (KO) mice using the CRISPR/Cas9 system. Sex determination and fetal testis development were apparently normal in mutant mice. Fertility analysis showed a low fecundity in Rhox8 KO adult males, with disrupted spermatogenic cycles, increased germ cell apoptosis, and reduced sperm count and motility. Interestingly, Rhox8 KO testes showed an increase in testis size with dilated seminiferous tubules and rete testis, which might be affected by efferent duct (ED) Rhox8 ablation dysregulating the expression of metabolism and transport genes in the EDs. Taken together, the data presented in this study suggest that Rhox8 in the Sertoli cells is not essential for sex determination and embryonic testis differentiation but has an important role in complete spermatogenesis and optimal male fertility.

Summary Sentence

Rhox8 knockout results in male subfertility together with a disrupted spermatogenic cycle, increased germ cell apoptosis, and reduced sperm count and motility, suggesting that Rhox8 has an important role for optimal male fertility.

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Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

Summary sentence

The Spry4 is a novel ERK-dependent negative feedback regulator of FGF and GDNF signaling that is critical for preservation of stem cell activity in cultured adult mouse spermatogonia and serves as a marker for adult SSCs in vivo.

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Exposure to phthalates disrupts ovarian function. However, limited studies have investigated the effects of phthalate mixtures on ovulation, especially in women. Human granulosa cells were used to test the hypothesis that exposure to a phthalate mixture (PHTmix) disrupts progesterone (P4)/progesterone receptor (PGR) signaling, which is a crucial pathway for ovulation. In addition, progestin and cyclic adenosine 3′, 5′-monophosphate (cAMP) supplementation were tested as methods to circumvent phthalate toxicity. Granulosa cells from women undergoing in vitro fertilization were acclimated in culture to regain responsiveness to human chorionic gonadotropin (hCG; clinical luteinizing hormone analogue). Granulosa cells were treated with or without hCG, and with or without PHTmix (1–500 µg/ml; dimethylsulfoxide = vehicle control) for 0.5–36 h. In the supplementation experiments, cells were treated with or without R5020 (stable progestin), and with or without 8-Br-cAMP (stable cAMP analogue). Exposure to hCG + PHTmix decreased P4 levels and mRNA levels of steroidogenic factors when compared to hCG. This was accompanied by decreased mRNA levels of PGR and downstream P4/PGR ovulatory mediators (ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), C-X-C motif chemokine receptor 4 (CXCR4), pentraxin 3 (PTX3), and regulator of G protein signaling 2 (RGS2)) in the hCG + PHTmix groups compared to hCG. Exposure to hCG + PHTmix 500 µg/ml decreased cAMP levels and protein kinase A activity compared to hCG. Supplementation with progestin in the hCG + PHTmix 500 µg/ml group did not rescue toxicity, while supplementation with cAMP restored PGR levels and downstream P4/PGR mediator levels to hCG levels. These findings suggest that phthalate mixture exposure inhibits P4/PGR signaling in human granulosa cells via decreased steroidogenesis, cAMP levels, and protein kinase A activity. Restored P4/PGR signaling with cAMP supplementation provides a potential cellular target for intervention of phthalate-induced ovulatory dysfunction in women.

Summary Sentence

Exposure to a phthalate mixture decreased progesterone production and inhibited PGR signaling in human granulosa cells, and PGR signaling was restored with cAMP supplementation.

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