Recurrent spontaneous abortion (RSA) is one of the major pregnancy disorders and poses a serious risk to both the mother and the fetus. Although a number of research efforts have been conducted, therapeutic advances for treating RSA have not lived up to their expectations. Hence, other treatments should be explored. The important role of natural killer (NK) cells in immunotherapy is attracting increasing attention, both as a pharmaceutical target and for cell therapies. NK cells are abundant in the endometrium and play a role in implantation and placentation in normal pregnancy. As research progresses, NK cells are increasingly regarded as playing essential roles in the emergence and development of RSA. In this article, I review recent findings on the role of uterine NK cells in the pathophysiology of RSA. These cells may become therapeutic NK cell-related targets. In conclusion, although several issues regarding NK cells in RSA remain unresolved and require further investigation, extensive evidence is available for the treatment of RSA.

Summary Sentence

In this review, we focus on recent research on the features and function of NK cell subsets, particularly decidual NK cells in maternal-fetal interface and their potential role in RSA.

The placenta is a transient organ but essential for the survival of all mammalian species by allowing for the exchanges of gasses, nutrients, and waste between maternal and fetal placenta. In rodents and humans with a hemochorial placenta, fetal placenta cells are susceptible to pharmaceutical agents and other compounds, as they are bathed directly in maternal blood. The placenta of mice and humans produce high concentrations of serotonin (5-HT) that can induce autocrine and paracrine effects within this organ. Placental 5-HT is the primary source of this neurotransmitter for fetal brain development. Increasing number of pregnant women at risk of depression are being treated with selective serotonin-reuptake inhibitors (SSRIs) that bind to serotonin transporters (SERT), which prevents 5-HT binding and cellular internalization, allowing for accumulation of extracellular 5-HT available to bind to 5-HT(2A) receptor (5-HT(2A)R). In vitro and in vivo findings with SSRI or pharmacological blockage of the 5-HT(2A)R reveal disruptions of 5-HT signaling within the placenta can affect cell proliferation, division, and invasion. In SERT knockout mice, numerous apoptotic trophoblast cells are observed, as well as extensive pathological changes within the junctional zone. Collective data suggest a fine equilibrium in 5-HT signaling is essential for maintaining normal placental structure and function. Deficiencies in placental 5-HT may also result in neurobehavioral abnormalities. Evidence supporting 5-HT production and signaling within the placenta will be reviewed. We will consider whether placental hyposerotonemia or hyperserotonemia results in similar pathophysiological changes in the placenta and other organs. Lastly, open ended questions and future directions will be explored.

Summary sentence

The fetal placenta produces serotonin (5-HT) that can induce autocrine/paracrine effects, but disruptions in this signaling pathway may lead to placental pathological changes and increase risk for fetal diseases, including autism spectrum disorders.

In Siberian hamsters, exposure to short days (SDs, 8 h light:16 h dark) reduces reproductive function centrally by decreasing gonadotropin secretion, whereas subsequent transfer of photoinhibited hamsters to stimulatory long days (LDs, 16 L:8 D) promotes follicle stimulating hormone (FSH) release inducing ovarian recrudescence. Although differences between SD and LD ovaries have been investigated, a systematic investigation of the ovarian transcriptome across photoperiod groups to identify potentially novel factors that contribute to photostimulated restoration of ovarian function had not been conducted. Hamsters were assigned to one of four photoperiod groups: LD to maintain ovarian cyclicity, SD to induce ovarian regression, or post transfer (PT), where females housed in SD for 14-weeks were transferred to LD for 2-days or 1-week to reflect photostimulated ovaries prior to (PTd2) and following (PTw1) the return of systemic FSH. Ovarian RNA was extracted to create RNA-sequencing libraries and short-read sequencing Illumina assays that mapped and quantified the ovarian transcriptomes (n = 4/group). Ovarian and uterine masses, plasma FSH, and numbers of antral follicles and corpora lutea decreased in SD as compared to LD ovaries (P < 0.05). When reads were aligned to the mouse genome, 18 548 genes were sufficiently quantified. Most of the differentially expressed genes noted between functional LD ovaries and regressed SD ovaries; however, five main expression patterns were identified across photoperiod groups. These results, generally corroborated by select protein immunostaining, provide a map of photoregulated ovary function and identify novel genes that may contribute to the photostimulated resumption of ovarian activity.

Summary sentence

Changes in photoperiod that induce ovarian regression and recrudescence or maintain ovarian function alter ovarian transcriptomes in Siberian hamsters.

The pregnancy complication preeclampsia is directly associated with hypoxic stress and insufficient trophoblast cell differentiation. The hypoxia-inducible microRNA (miRNA), miR-210, has been identified as a significantly up-regulated miRNA in preeclamptic placenta, and evidence in other cell types has indicated a feedback regulation between miR-210 and hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. It remains unclear whether and how the feedback loop between miR-210 and HIF-1α may contribute to trophoblast dysfunction in preeclampsia. Here, we proved that cytoplasmic polyadenylation element-binding 2 (CPEB2) was a direct target of miR-210 in human trophoblast. CPEB2 could inhibit the translation of hypoxia-induced HIF-1α via directly binding the cytoplasmic polyadenylation element (CPE) site in the 3′-untranslated region (UTR) of HIF-1α mRNA. The increase in the HIF-1α level upon hypoxia treatment could be efficiently reversed by miR-210 inhibitor. In addition, CPEB2 was primarily expressed in villous syncytiotrophoblasts, and the suppression of trophoblast cell syncytialization by miR-210 could be significantly rescued by CPEB2 overexpression. In preeclamptic placenta, the expression of CPEB2 was evidently lower than normal pregnant control, and the miR-210 level was aberrantly higher and trophoblast syncytialization was limited. The findings revealed a positive feedback loop between miR-210 and HIF-1α that is mediated by CPEB2 in human trophoblasts, and demonstrated a mechanism underlying the insufficient trophoblast syncytialization in preeclampsia under hypoxic stress.

Summary sentence

A positive feedback loop between miR-210 and HIF-1α that is mediated by CPEB2 in human trophoblast demonstrates the mechanism of insufficient syncytialization in preeclampsia under hypoxic stress.

Survival and growth of the bovine conceptus is dependent on endometrial secretions or histotroph. Previously, serial blastocyst transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components (proteins and metabolites) in the uterine lumen of day 17 fertility-classified heifers. Interferon tau (IFNT) was more abundant in uterine lumenal fluid (ULF) of pregnant HF than SF animals as the conceptus was longer in HF heifers. However, no differences in endometrial expression of selected classical and nonclassical interferon-stimulated genes (ISGs) were observed, suggesting that IFNT signaling in the endometrium of pregnant HF and SF heifers was similar. Pregnancy significantly increased the abundance of several proteins in ULF. Based on functional annotation, the abundance of a number of proteins involved in energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were greater in the ULF of pregnant HF than SF heifers. Metabolomics analysis found that pregnancy only changed the metabolome composition of ULF from HF heifers. The majority of the metabolites that increased in the ULF of pregnant HF as compared to SF heifers were associated with energy and amino acid metabolism. The observed differences in ULF proteome and metabolome are hypothesized to influence uterine receptivity with consequences on conceptus development and survival in fertility-classified heifers.

Summary sentence

Pregnancy induced changes in the uterine lumen are dysregulated in subfertile heifers.

The Sertoli cell (Sc) has been described as a quiescent cell once the animal has reached sexual maturity. Syrian hamster is an animal that displays testicular regression due to short photoperiod, during which process germ cells and Sc are removed through apoptosis. The aim of this work was to investigate histochemically whether the spontaneous testicular recrudescence processes after exposure to a short photoperiod lead to an increase in Sc proliferative activity in order to restore the normal population. Three spontaneous recrudescence groups were established: initial (IR), advanced (AR), and total (TR) recrudescence, which were compared with animal undergoing the regression process (mild: MRg, strong: SRg, and total: TRg) and animals in long photoperiod (Controls). Histological sections were submitted to histochemical techniques for detecting apoptotic and proliferative Sc with bright-field and fluorescence microscopy. For each group, the proliferative Sc index (PScI) and apoptotic Sc index (AScI), and the total number of Sc were obtained. The results revealed the existence of Vimentin+/TUNEL+ as well as Vimentin+/PCNA+ cells. The PScI was significantly higher in TRg and IR than in the other groups. The AScI was only significantly higher in MRg and SRg with respect to the other groups. The total number of Sc increased among TRg, IR, and AR, reaching values similar to those of the Controls. In conclusion, the increase in Sc proliferation from final regression and recrudescence, accompanied by a similar rate of apoptosis to the Control group, is the cause of the restoration of the Sc population during spontaneous recrudescence.

Summary Sentence

The total population of Scs is recovered after spontaneous recrudescence due to an increase in Sc proliferation.

The placenta, which originates from the trophectoderm (TE), is the first organ to form during mammalian embryogenesis. Recent studies based on bioinformatics analysis have revealed that heterogeneous gene expression initiates cell-fate decisions and directs two distinct cell fates by modulating the balance of pluripotency and differentiation as early as the four-cell stage. However, direct developmental evidence to support this is still lacking. To address at which stage the cell fate of the TE and inner cell mass (ICM) is determined, in this study, we administered a microinjection of Cre mRNA into a single blastomere of the mTmG mouse at different cleavage stages before implantation to examine the distributions of the descendants of the single-labeled cell in the mouse fetus and the placenta at E12.5. We found that the descendants of the labeled cells at the two-cell stage contributed to both the placenta and the fetus. Notably, the derivatives of the labeled cells at the four-cell stage fell into three categories: (1) distributed in both embryonic and extraembryonic lineages, (2) distributed only in mouse placental trophoblast layers, or (3) distributed only in the lineage derived from the ICM. In addition, these results fell in line with single-cell studies focusing on gene expression patterns that characterize particular lineages within the blastocyst. In conclusion, this study shows that the four-cell blastomeres differ in their individual developmental properties insofar as they contribute to either or both the ICM and trophoblast fate.

Summary sentence

The four-cell blastomeres differ in their individual developmental properties insofar as they contribute to either or both the embryonic and extraembryonic lineage.

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

Summary sentence

Our studies show that high heteroplasmy deleterious mtDNA mutations presenting in mature mouse oocytes are eliminated during post-implantation development. The occurrence of purifying selection against deleterious mtDNA mutations during fetal development plays an important role in preventing the accumulation of pathogenic mutations that would cause consequences to species survival.

In vitro reconstitution of germ-cell development from pluripotent stem cells (PSCs) has created key opportunities to explore the fundamental mechanisms underlying germ-cell development, particularly in mice and humans. Importantly, such investigations have clarified critical species differences in the mechanisms regulating mouse and human germ-cell development, highlighting the necessity of establishing an in vitro germ-cell development system in other mammals, such as non-human primates. Here, we show that multiple lines of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in cynomolgus monkeys (Macaca fascicularis; cy) can be maintained stably in an undifferentiated state under a defined condition with an inhibitor for WNT signaling, and such PSCs are induced efficiently into primordial germ cell-like cells (PGCLCs) bearing a transcriptome similar to early cyPGCs. Interestingly, the induction kinetics of cyPGCLCs from cyPSCs is faster than that of human (h) PGCLCs from hPSCs, and while the transcriptome dynamics during cyPGCLC induction is relatively similar to that during hPGCLC induction, it is substantially divergent from that during mouse (m) PGCLC induction. Our findings delineate common as well as species-specific traits for PGC specification, creating a foundation for parallel investigations into the mechanism for germ-cell development in mice, monkeys, and humans.

Summary Sentence

Pluripotent stem cells in cynomolgus monkeys are induced into primordial germ cell-like cells with species-specific kinetics and transcriptome dynamics.

Activins selectively stimulate follicle-stimulating hormone (FSH) secretion by pituitary gonadotrope cells. More recently, other members of the TGFbeta superfamily, the bone morphogenetic proteins (BMPs), were reported to regulate FSH synthesis. Activins and BMPs independently and synergistically stimulate transcription of the FSHbeta subunit (Fshb) gene in immortalized gonadotrope-like cells. Both ligands can signal via the activin receptor type IIA (ACVR2A) to regulate FSH synthesis in vitro. In vivo, global Acvr2a knockout mice exhibit a 60% reduction in circulating FSH relative to wild-type animals, suggesting that activins, BMPs, or related ligands might signal through additional type II receptors to regulate FSH in vivo. Although the leading candidates are ACVR2B and the BMP type II receptor (BMPR2), only the latter mediates activin or BMP2 induction of Fshb transcription in vitro. Here, we generated mice carrying a loss of function mutation in Bmpr2 specifically in gonadotropes. Puberty onset, estrous cyclicity, and reproductive organ weights were similar between control and conditional knockout females. Serum FSH and luteinizing hormone (LH) and pituitary expression of Fshb and the LHbeta subunit (Lhb) were similarly unaffected by the gene deletion in both sexes. These results suggest that BMPR2 might not play a necessary role in FSH synthesis or secretion in vivo or that another type II receptor, such as ACVR2A, can fully compensate for its absence. These data also further contribute to the emerging concept that BMPs may not be physiological regulators of FSH in vivo.

Summary sentence

Ablation of BMPR2 in gonadotropes does not affect gonadotropin levels, suggesting that TGFbeta ligands other than BMPs and activins may be the primary regulators of FSH synthesis in mice.

Abstract Reproduction is a major component of an animal's life history strategy. Species with plasticity in their reproductive biology are likely to be successful as an invasive species, as they can adapt their reproductive effort during various phases of a biological invasion. Silver carp (Hypophthalmicthys molitrix), an invasive cyprinid in North America, display wide variation in reproductive strategies across both their native and introduced ranges, though the specifics of silver carp reproduction in the Illinois River have not been established. We assessed reproductive status using histological and endocrinological methods in silver carp between April and October 2018, with additional histological data from August to October 2017. Here, we show that female silver carp are batch spawners with asynchronous, indeterminate oocyte recruitment, while male silver carp utilize a determinate pattern of spermatogenesis which ceases in the early summer. High plasma testosterone levels in females could be responsible for regulating oocyte development. Our results suggest that silver carp have high spawning activity in the early summer (May–June), but outside of the peak spawning period, female silver carp can maintain spawning-capable status by adjusting rates of gametogenesis and atresia in response to environmental conditions, while males regress their gonads as early as July. The results of this study are compared to reports of silver carp reproduction in other North American rivers as well as in Asia.

Incomplete maternal vascular responses to pregnancy contribute to pregnancy complications including intrauterine growth restriction (IUGR) and preeclampsia. We aimed to characterize maternal vascular dysfunction in a murine model of fetal growth restriction as an approach toward identifying targetable pathways for improving pregnancy outcomes. We utilized a murine model of late-gestation hypoxia-induced IUGR that reduced E18.5 fetal weight by 34%. Contrary to our hypothesis, uterine artery blood flow as measured in vivo by Doppler ultrasound was increased in mice housed under hypobaric hypoxia (385 mmHg; 5500 m) vs normoxia (760 mmHg; 0 m). Using wire myography, uterine arteries isolated from hypoxic mice had similar vasodilator responses to the two activators A769662 and acetylcholine as those from normoxic mice, although the contribution of an increase in nitric oxide production to uterine artery vasodilation was reduced in the hypoxic vs normoxic groups. Vasoconstrictor responses to phenylephrine and potassium chloride were unaltered by hypoxia. The levels of activated adenosine monophosphate-activated protein kinase (AMPK) were reduced with hypoxia in both the uterine artery and placenta as measured by western blot and immunohistochemistry. We concluded that the rise in uterine artery blood flow may be compensatory to hypoxia but was not sufficient to prevent fetal growth restriction. Although AMPK signaling was reduced by hypoxia, AMPK was still receptive to pharmacologic activation in the uterine arteries in which it was a potent vasodilator. Thus, AMPK activation may represent a new therapy for pregnancy complications involving reduced uteroplacental perfusion.

Summary sentence

Late-gestation hypoxia in murine pregnancy induces fetal growth restriction while raising uterine artery volumetric blood flow and reducing uteroplacental adenosine monophosphate-activated protein kinase (AMPK) activation.

Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.

Summary sentence

RNAseq coupled with transcriptional inhibition suggests that gene expression is critical for degradation of maternal mRNAs and that KLF family members regulate expression of ∼ 50% of embryonically expressed genes.

Follicular progression during peripuberty is affected by diet. Vascular endothelial growth factor A (VEGFA) induces follicle progression in many species; however, there are limited studies to determine if diet may alter the effects of angiogenic VEGFA165-stimulated follicle progression or antiangiogenic VEGFA165b follicle arrest. We hypothesized that diet affects the magnitude of angiogenic and antiangiogenic VEGFA isoform actions on follicular development through diverse signal transduction pathways. To test this hypothesis, beef heifers in our first trial received Stair-Step (restricted and refeeding) or control diets from 8 to 13 months of age. Ovaries were collected to determine follicle stages, measure vascular gene expression and conduct ovarian cortical cultures. Ovarian cortical cultures were treated with phosphate-buffered saline (control), 50 ng/ml VEGFA165, VEGFA165b, or VEGFA165 + VEGFA165b. The Stair-Step heifers had more primordial follicles (P < 0.0001), greater messenger RNA abundance of vascular markers VE-cadherin (P < 0.0001) and NRP-1 (P < 0.0051) than controls at 13 months of age prior to culture. After culture, VEGFA isoforms had similar effects, independent of diet, where VEGFA165 stimulated and VEGFA165b inhibited VEGFA165-stimulated follicle progression from early primary to antral follicle stages. In vitro cultures were treated with VEGFA isoforms and signal transduction array plates were evaluated. VEGFA165 stimulated expression of genes related to cell cycle, cell proliferation, and growth while VEGFA165b inhibited expression of those genes. Thus, VEGFA isoforms can act independently of diet to alter follicle progression or arrest. Furthermore, follicle progression can be stimulated by VEGFA165 and inhibited by VEGFA165b through diverse signal transduction pathways.

Summary Sentence

Prepubertal in vivo diets do not affect in vitro VEGFA isoforms ability to inhibit or progress follicles by diverse signal transduction pathways in bovine cortex tissue.

Bisphenol A (BPA) is commonly found in epoxy resins used in the manufacture of plastic coatings in food packaging and beverage cans. There is a growing concern about BPA as a weak estrogenic compound that can affect human endocrine function. Chemicals structurally similar to BPA, such as bisphenol F (BPF) and bisphenol S (BPS), have been developed as substitutes in the manufacturing industry. Whether these bisphenol substitutes have adverse effects on human endocrine and reproductive systems remains largely unknown. This study investigated the effects of BPA, BPF, and BPS on regulating the function of decidualized human primary endometrial stromal cells on trophoblast outgrowth and invasion by indirect and direct co-culture models. All three bisphenols did not affect the stromal cell decidualization process. However, BPA- and BPF-treated decidualized stromal cells stimulated trophoblastic spheroid invasion in the indirect coculture model. The BPA-treated decidualized stromal cells had upregulated expressions of several invasion-related molecules including leukemia inhibitory factor (LIF), whereas both BPA- and BPF-treated decidualized stromal cells had downregulated expressions of anti-invasion molecules including plasminogen activator inhibitor type 1 (PAI-1) and tumor necrosis factor (TNFα) . Taken together, BPA and BPF altered the expression of invasive and anti-invasive molecules in decidualized stromal cells modulating its function on trophoblast outgrowth and invasion, which could affect the implantation process and subsequent pregnancy outcome.

Maternal high-fat (HF) diet negatively affects maternal metabolism and placental function. This study aimed to determine whether gestational exercise prevents the effect of HF diet on placental amino acid transporter expression and nutrient-sensing signaling and the fetal response. Pregnant Sprague-Dawley rats were either fed with a CHOW (13.5% fat) or HF (60% fat) diet during gestation and further divided into two subgroups: voluntary exercised and sedentary. Placentae were collected on gestational day (GD) 14 and GD20, and male placentae were used in this study. We found that gestational exercise ameliorated the detrimental effects of HF diet on dams' adiposity, plasma leptin, and insulin concentrations. Maternal exercise did not influence fetoplacental growth but affected male fetal hypothalamic Leprb, Stat3, Insr, Agrp, and Pomc expressions on GD20. Maternal HF diet decreased placental labyrinth thickness and increased system A amino acid transporter SNAT2 expression, while these changes were normalized by exercise. The activation of placental mechanistic target of rapamycin complex 1/4E-BP1 and LepRb/STAT3 signaling might contribute to the increased placental SNAT2 expression in HF-fed dams, which were reversed by exercise on GD20. These data highlight that gestational exercise reverses HF-diet-induced placental alterations during late gestation without influencing fetal growth. However, maternal exercise altered fetal hypothalamic gene expression, which may affect long-term offspring health.

Summary sentence

Gestational exercise reverses high-fat diet induced placental alterations during late gestation without influencing fetal growth, but altered male fetal hypothalamic gene expression.

Understanding and control of the massive and accelerated follicular growth that occurs during in vitro culture of ovarian tissue is a crucial step toward the development of efficient culture systems that offer an attractive alternative to ovarian tissue transplantation for fertility restoration in cancer survivors. One outstanding question focuses on processes that occur prior to cryopreservation, such as tissue sectioning or chemotherapeutic treatment, might exacerbate this follicular activation. Although the PI3K/AKT/mTOR pathway is well known as a major trigger of physiological and chemotherapy-induced follicular activation, studies have shown that disruption of Hippo pathway due to ovarian fragmentation acts as an additional stimulator. This study aimed to characterize the possible interactions between these pathways using post-natal day 3 mouse ovaries cultured for 4 or 48 h. Morphology, gene transcription, and protein levels were assessed to investigate the impact of sectioning or chemotherapy exposure (4-hydroperoxycyclophosphamide [4HC], 3 and 20 µM). The effect of an mTORC1 inhibitor, Everolimus, alone or as a 4HC co-treatment to prevent follicle activation was evaluated. The results showed that organ removal from its physiological environment was as effective as sectioning for disruption of Hippo pathway and induction of follicle activation. Both PI3K/AKT/mTOR and Hippo pathways were involved in chemotherapy-induced follicular activation and responded to fragmentation. Surprisingly, Everolimus was able to prevent the activation of both pathways during chemotherapy exposure, suggesting cross-talk between them. This study underscores the major involvement of PI3K/AKT/mTOR and Hippo pathways in in vitro follicle activation and provides evidence that both can be regulated using mTORC1 inhibitor.

Summary sentence

PI3K and Hippo pathways interact to trigger spontaneous and induced in vitro follicular activation and mTORC1 inhibitor acts on both of them to moderate their effects.

Over the last decades, fertility of dairy cows has declined due to selection strategies focusing on milk yield. To study the effect of genetic merit for fertility on the proteome of the bovine uterine luminal fluid, Holstein heifers with low- and two groups of heifers with high-fertility index (high-fertility Holstein and Montbéliarde) were investigated. To focus on the maternal effect, heifers from all groups were synchronized and received on Day 7 high-quality embryos. Uterine luminal fluid from Day 19 pregnant heifers was analyzed in a holistic proteomic approach using nano-LC-MS/MS analysis combined with a label-free quantification approach. In total, 1737 proteins were identified, of which 597 differed significantly in abundance between the three groups. The vast majority of proteome differences was found comparing both high-fertility groups to the low-fertility Holstein group, showing that the genetic predisposition for fertility is prevalent regarding the uterine luminal fluid proteome. Evaluation of this dataset using bioinformatic tools revealed an assignment of higher abundant proteins in low-fertility Holstein to several metabolic processes, such as vitamin metabolic process, which comprises folate receptor alpha (FOLR1) and retinol-binding protein, indicating an involvement of disturbed metabolic processes in decreased fertility. Moreover, immune system-related proteins — lactotransferrin and chromogranin A — were enriched in low-fertility cows together with interferon tau 3 h and interferon tau-2. Our results indicate that the genetic merit for fertility leads to substantial quantitative differences at the level of proteins in uterine fluid of pregnant animals, thus altering the microenvironment for the early conceptus.

Summary sentence

The proteome of bovine uterine fluid, which is crucial for conceptus development prior to implantation, is significantly altered by the genetic merit for fertility.

Identification of reliable characteristics of follicle quality and developmental competence has been pursued in numerous studies, but with inconsistent outcomes. Here, we aimed to identify these characteristics by analysis of the follicular fluid (FF) steroid profile in relation to cumulus-oocyte complex (COC) morphology and follicle size, followed by molecular substantiation. Multiparous sows at weaning were used to facilitate analysis at the start of the follicular phase of the oestrus cycle. Sows with a higher average follicle size (≥5 mm vs. < 5 mm) had a higher follicular fluid β-estradiol concentration, but did not differ in other measured steroids. Sows with high compared to low percentage high-quality COCs (<70% vs. ≥70% high-quality) had follicular fluid with a higher concentration of β-estradiol, 19-norandrostenedione, progesterone, and α-testosterone, while the concentration of cortisol was lower. Transcriptome analysis of granulosa cells of healthy follicles of sows with a high percentage high-quality COCs showed higher abundance of transcripts involved in ovarian steroidogenesis (e.g., CYP19A2 and 3, POR, VEGFA) and growth (IGF1) and differential abundance of transcripts involved in granulosa cell apoptosis (e.g., GADD45A, INHBB). Differences in aromatase transcript abundance (CYP19A1, 2 and 3) were confirmed at the protein level. In addition, sows with a high percentage high-quality COCs lost less weight during lactation and had higher plasma IGF1 concentration at weaning, which may have affected COC quality. To the best of our knowledge, this study is also the first to report the relation between FF steroid profile and COC quality.

Summary sentence

Sows with a higher percentage high-quality COCs have a distinct follicular fluid steroid profile and concomitant granulosa cell transcriptome.

The acrosome reaction is a multi-step event essential for physiological fertilization. During the acrosome reaction, gamete fusion-related factor IZUMO1 translocates from the anterior acrosome to the equatorial segment and assembles the gamete fusion machinery. The morphological changes in the acrosome reaction process have been well studied, but little is known about the molecular mechanisms of acrosome reorganization essential for physiological gamete membrane fusion. To elucidate the molecular mechanisms of IZUMO1 translocation, the steps of the acrosome reaction during that process must be clarified. In this study, we established a method to detect the early steps of the acrosome reaction and subdivided the process into seven populations through the use of two epitope-defined antibodies, anti-IZUMO1 and anti-SPACA1, a fertilization-inhibiting antibody. We found that part of the SPACA1 C-terminus in the periacrosomal space was cleaved and had begun to disappear when the vesiculation of the anterior acrosome occurred. The IZUMO1 epitope externalized from the acrosomal lumen before acrosomal vesiculation and phosphorylation of IZUMO1 occurred during the translocation to the equatorial segment. IZUMO1 circumvented the area of the equatorial segment where the SPACA1C-terminus was still localized. We therefore propose an IZUMO1 translocation model and involvement of SPACA1.

Summary sentence

Cleavage of SPACA1 regulates the translocation of IZUMO1 to form gamete membrane fusion machinery on the equatorial segment.

Foxh1, a member of fox gene family, was first characterized as a transcriptional partner in the formation of the Smad protein complex. Recent studies have shown foxh1 is highly expressed in the cytoplasm of oocytes in both tilapia and mouse. However, its function in oogenesis remains unexplored. In the present study, foxh1^–/– tilapia was created by CRISPR/Cas9. At 180 dah (days after hatching), the foxh1^–/– XX fish showed oogenesis arrest and a significantly lower GSI. The transition of oocytes from phase II to phase III and follicle cells from one to two layers was blocked, resulting in infertility of the mutant. Transcriptomic analysis revealed that expression of genes involved in estrogen synthesis and oocyte growth were altered in the foxh1^–/– ovaries. Loss of foxh1 resulted in significantly decreased Cyp19a1a and increased Cyp11b2 expression, consistent with significantly lower concentrations of serum estradiol-17β (E2) and higher concentrations of 11-ketotestosterone (11-KT). Moreover, administration of E2 rescued the phenotypes of foxh1^–/– XX fish, as indicated by the appearance of phase III and IV oocytes and absence of Cyp11b2 expression. Taken together, these results suggest that foxh1 functions in the oocytes to regulate oogenesis by promoting cyp19a1a expression, and therefore estrogen production. Disruption of foxh1 may block the estrogen synthesis and oocyte growth, leading to the arrest of oogenesis and thus infertility in tilapia.