Reprogramming of histone modifications is critical to safeguard correct gene expression profile during preimplantation development. Of interest, trimethylation of lysine 4 on histone 3 (H3K4me3) exhibits a unique and dynamic landscape with a potential species-specific feature. Here, we address how it is reprogrammed and its functional significance during oocyte maturation and early embryonic development in cows. Notably, the overall signal of H3K4me3 decreased sharply during embryonic genome activation (EGA). By using low input ChIP-seq, we find widespread broad H3K4me3 domains in oocytes and early cleaved embryos. The broad domains are gradually removed after fertilization, which is obviously seen during EGA. Meanwhile, H3K4me3 becomes enriched at promoter regions after the removal of broad H3K4me3. Interestingly, the gene expression level displays a positive correlation with the relative H3K4me3 signal of their promoters when embryos reach 16-cell stage. Importantly, disruption of KDM5 (H3K4me3 demethylases) increases H3K4me3 level, decreases the embryonic developmental rate, and results in dysregulation of over a thousand genes. Meanwhile, KDM5 deficiency causes a redistribution of H3K4me3 across genome. In particular, H3K4me3 in gene body or intergenic regions cannot be removed, and H3K4me3 in promoter regions is aberrantly reduced. Besides, the positive correlation between promoter H3K4me3 enrichment and gene expression level disappears. Overall, we describe the genomic reprogramming of H3K4me3 with a greater resolution during bovine preimplantation development and propose that KDM5-mediated redistribution of H3K4me3 plays an important role in modulating oocyte-to-embryonic transition.

Summary Sentence

KDM5-mediated redistribution of H3K4me3 plays an important role in modulating oocyte-to-embryonic transition.

Sirtuin 1 (SIRT1) is a member of the sirtuin family that functions to deacetylate both histones and non-histone proteins. Previous studies have identified significant SIRT1 upregulation in eutopic endometrium from infertile women with endometriosis. However, SIRT1 function in the uterus has not been directly studied. Using immunochemistry analysis, we found SIRT1 to be most strongly expressed at GD4.5 and GD5.5 in decidualized cells and at GD7.5 in secondary decidual cells in mouse. To assess the role of SIRT1 in uterine function, we generated uterine Sirt1 conditional knockout mice (Pgr^cre/+Sirt1^f/f; Sirt1^d/d). A 6-month fertility trial revealed that Sirt1^d/d females were subfertile. Implantation site numbers were significantly decreased in Sirt1^d/d mice compared with controls at GD5.5. Sirt1^d/d implantation sites at GD4.5 could be divided into two groups, Group #1 with luminal closure and nonspecific COX2 expression compared with controls (14/20) and Group #2 with an open lumen and no COX2 (6/20). In Sirt1^d/d Group #1, nuclear FOXO1 expression in luminal epithelial cells was significantly decreased. In Sirt1^d/d Group #2, nuclear FOXO1 expression was almost completely absent, and there was strong PGR expression in epithelial cells. At GD5.5, stromal PGR and COX2 were significantly decreased in Sirt1^d/d uterine in the areas surrounding the embryo compared with controls, indicating defective decidualization. An artificially induced decidualization test revealed that Sirt1^d/d females showed defects in decidualization response. All together, these data suggest that SIRT1 is important for decidualization and contributes to preparing a receptive endometrium for successful implantation.

Bone marrow-derived progenitor cells (BMDPCs) are mobilized to the circulation in pregnancy and get recruited to the pregnant decidua where they contribute functionally to decidualization and successful implantation. However, the molecular mechanisms underlying BMDPCs recruitment to the decidua are unknown. CXCL12 ligand and its CXCR4 receptor play crucial roles in the mobilization and homing of stem/progenitor cells to various tissues. To investigate the role of CXCL12–CXCR4 axis in BMDPCs recruitment to decidua, we created transgenic GFP mice harboring CXCR4 gene susceptible to tamoxifen-inducible Cre-mediated ablation. These mice served as BM donors into wild-type C57BL/6 J female recipients using a 5-fluorouracil-based nongonadotoxic submyeloablation to achieve BM-specific CXCR4 knockout (CXCR4KO). Successful CXCR4 ablation was confirmed by RT-PCR and in vitro cell migration assays. Flow cytometry and immunohistochemistry showed a significant increase in GFP+ BM-derived cells (BMDCs) in the implantation site as compared to the nonpregnant uterus of control (2.7-fold) and CXCR4KO (1.8-fold) mice. This increase was uterus-specific and was not observed in other organs. This pregnancy-induced increase occurred in both hematopoietic (CD45+) and nonhematopoietic (CD45–) uterine BMDCs in control mice. In contrast, in CXCR4KO mice there was no increase in nonhematopoietic BMDCs in the pregnant uterus. Moreover, decidual recruitment of myeloid cells but not NK cells was diminished by BM CXCR4 deletion. Immunofluorescence showed the presence of nonhematopoietic GFP+ cells that were negative for CD45 (panleukocyte) and DBA (NK) markers in control but not CXCR4KO decidua. In conclusion, we report that CXCR4 expression in nonhematopoietic BMDPCs is essential for their recruitment to the pregnant decidua.

Summary statement: Expression of CXCR4 receptor in nonhematopoietic bone marrow-derived progenitor cells is essential for their recruitment to the decidua during pregnancy.

Graphical Abstract

Among the many calcium-binding proteins, S100A8, S100A9, and S100A12 play important roles in inflammation, innate immunity, and antimicrobial function, but their expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression and regulation of S100A8, S100A9, S100A12, and their receptor AGER at the maternal-conceptus interface in pigs. We found that S100A8, S100A9, and S100A12 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy, with the greatest levels on Day (D) 12 of pregnancy, and AGER appeared at greater levels on D15 and D30 of pregnancy than on other days. The expression of S100A8, S100A9, and S100A12 was predominantly localized to epithelial cells in the endometrium, and they were detected in early-stage conceptus and later chorioallantoic tissues during pregnancy. AGER expression was localized to endometrial epithelial and stromal cells and chorionic epithelial cells. In endometrial explant tissues, the expression of S100A8, S100A9, and S100A12 was induced by estrogen, S100A8 by interleukin-1β, and AGER by interferon-γ . We further found that on D12 of pregnancy, the expression of S100A8, S100A9, and S100A12 decreased significantly in the endometria of gilts carrying conceptuses derived from somatic cell nuclear transfer. These results indicate that the expression of S100A8, S100A9, and S100A12 is dynamically regulated in response to conceptus-derived signals at the maternal-conceptus interface, suggesting that S100A8, S100A9, and S100A12 could play a critical role in regulating endometrial epithelial cell function and conceptus implantation to support the establishment and maintenance of pregnancy in pigs.

Summary Sentence

S100A calcium-binding proteins S100A8, S100A9, and S100A12 are dynamically expressed in response to conceptus-derived signals at the maternal-conceptus interface in pigs.

Endometrial receptivity damage caused by impaired decidualization may be one of the mechanisms of infertility in endometriosis (EMs). Our previous study demonstrated that Calpain-7 (CAPN7) is abnormally overexpressed in EMs. Whether CAPN7 affects the regulation of decidualization and by what mechanism CAPN7 regulates decidualization remains to be determined. In this study, we found CAPN7 expression decreased during human endometrial stromal cell (HESC) decidualization in vitro. CAPN7 negatively regulated decidualization in vitro and in vivo. We also identified one conserved potential PEST sequence in the AKT1 protein and found that CAPN7 was able to hydrolyse AKT1 and enhance AKT1's phosphorylation. Correspondingly, CAPN7 notably promoted the phosphorylation of Forkhead Box O1 (FoxO1), the downstream of AKT1 protein, at Ser319, leading to increased FoxO1 exclusion from nuclei and attenuated FoxO1 transcriptional activity in decidualized HESC. In addition, we detected endometrium CAPN7, p-AKT1, and p-FoxO1 expressions were increased in EMs. These data demonstrate that CAPN7 negatively regulates HESC decidualization in EMs probably by promoting FoxO1's phosphorylation and FoxO1 nuclear exclusion via hydrolyzing AKT1. The dysregulation of CAPN7 may be a novel cause of EMs.

This study aimed to determine whether the acceleration of conceptus development induced by the administration of exogenous progesterone (P4) during the preimplantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal–conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil (CO) vehicle or 25 mg of progesterone in CO (P4) for the first 8 days of pregnancy and hysterectomized on either Day 9 (CO, n = 5; P4, n = 6), 12 (CO, n = 9; P4, n = 4) or 125 (CO, n = 14; P4, n = 10) of gestation. The expression of S100A12 (P < 0.05) and fibroblast growth factor receptor (FGFR2) (P < 0.01) messenger RNAs (mRNAs) was lower in endometria from P4-treated ewes on Day 12. The expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. The expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), solute carrier (SLC)20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, the expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, vitamin D receptor (VDR), S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered the expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal–conceptus interface.

Summary Sentence

Exogenous progesterone in early pregnancy affects the expression of regulators of calcium, phosphate, and vitamin D in late pregnancy, suggesting previously unappreciated effects on mineral transport at the maternal–conceptus interface of sheep.

Podocalyxin (PODXL) is a newly identified key negative regulator of human endometrial receptivity, specifically down-regulated in the luminal epithelium at receptivity to permit embryo implantation. Here, we bioinformatically compared the molecular characteristics of PODXL among the human, rhesus macaque, and mouse, determined by immunohistochemistry and in situ hybridization (mouse tissues) whether endometrial PODXL expression is conserved across the three species and examined if PODXL inhibits mouse embryo attachment in vitro. The PODXL gene, mRNA, and protein sequences showed greater similarities between humans and macaques than with mice. In all species, PODXL was expressed in endometrial luminal/glandular epithelia and endothelia. In macaques (n = 9), luminal PODXL was significantly down-regulated when receptivity is developed, consistent with the pattern found in women. At receptivity, PODXL was also reduced in shallow glands, whereas endothelial expression was unchanged across the menstrual cycle. In mice, endometrial PODXL did not vary considerably across the estrous cycle (n = 16); however, around embryo attachment on d4.5 of pregnancy (n = 4), luminal PODXL was greatly reduced especially near the site of embryo attachment. Mouse embryos failed to attach or thrive when co-cultured on a monolayer of Ishikawa cells overexpressing PODXL. Thus, endometrial luminal PODXL expression is down-regulated for embryo implantation in all species examined, and PODXL inhibits mouse embryo implantation. Rhesus macaques share greater conservations with humans than mice in PODXL molecular characteristics and regulation, thus represent a better animal model for functional studies of endometrial PODXL for treatment of human fertility.

Summary Sentence

Podocalyxin, a key negative regulator of human endometrial receptivity, is also down-regulated in the luminal epithelium in both the rhesus macaque and mouse uterus for embryo implantation, and podocalyxin inhibits mouse embryo attachment in vitro.

The appropriate balance between pro-inflammatory and anti-inflammatory cytokines is important for the maternal immune tolerance during pregnancy in mammals. Among the various cytokines, interleukin (IL)-10 (IL10) plays an essential role in anti-inflammatory responses, while IL12 is involved in pro-inflammatory responses during pregnancy. However, the roles of IL10 and IL12 in the endometrium during pregnancy have not been studied in pigs. Thus, we investigated the expression of IL10, IL12 (IL12A and IL12B), and their receptors (IL10RA, IL10RB, IL12RB1, and IL12RB2) at the maternal–conceptus interface. IL10, IL12, and their receptors were expressed in the endometrium during the estrous cycle and pregnancy in a pregnancy stage-specific manner. During pregnancy, IL10 expression increased on Day 15, whereas the expression of IL12A and IL12B decreased after the implantation period. IL10 protein was localized to luminal epithelial (LE), stromal cells, and macrophages; IL10RA protein to LE, endothelial, stromal, and T cells; and IL10RB mRNA to LE cells in the endometrium. IL10 and IL10RA proteins and IL10RB mRNA were also localized to chorionic epithelial (CE) cells. In endometrial explants, the expression of IL10RA and IL10RB was induced by estradiol-17β, IL-1β, and/or interferon-γ . Heme oxygenase 1, an IL10-inducible factor, was expressed in the endometrium with the highest levels on Day 30 of pregnancy and was localized to LE and CE cells. These results in pigs suggest that conceptus-derived signals change the endometrial immune environment by regulating the expression of IL10 and IL10 receptors at the maternal–conceptus interface and that IL10 may provide anti-inflammatory conditions for the maternal immune tolerance.

Summary Sentence

IL10 expression increases at the maternal–conceptus interface in pigs.

Spermatogenic regeneration is key for male fertility and relies on activities of an undifferentiated spermatogonial population. Here, a high-throughput approach with primary cultures of mouse spermatogonia was devised to rapidly predict alterations in functional capacity. Combining the platform with a large-scale RNAi screen of transcription factors, we generated a repository of new information from which pathway analysis was able to predict candidate molecular networks regulating regenerative functions. Extending from this database, the SRCAP-CREBBP/EP300 (Snf2-related CREBBP activator protein-CREB binding protein/E1A binding protein P300) complex was found to mediate differential levels of histone acetylation between stem cell and progenitor spermatogonia to influence expression of key self-renewal genes including the previously undescribed testis-specific transcription factor ZSCAN2 (zinc finger and SCAN domain containing 2). Single cell RNA sequencing analysis revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. In Zscan2 knockout mice, while spermatogenesis was moderately impacted during steady state, regeneration after cytotoxic insult was significantly impaired. Altogether, these findings have validated the utility of our high-throughput screening approach and have generated a transcription factor database that can be utilized for uncovering novel mechanisms governing spermatogonial functions.

Summary Sentence

A high-throughput RNAi screen was conducted to identify candidate molecules and molecular networks that control the regenerative function of spermatogonial stem cells.

Members of the nuclear factor I (NFI) family are key regulators of stem cell biology during development, with well-documented roles for NFIA, NFIB, and NFIX in a variety of developing tissues, including brain, muscle, and lung. Given the central role these factors play in stem cell biology, we posited that they may be pivotal for spermatogonial stem cells or further developing spermatogonia during testicular development. Surprisingly, in stark contrast to other developing organ systems where NFI members are co-expressed, these NFI family members show discrete patterns of expression within the seminiferous tubules. Sertoli cells (spermatogenic supporting cells) express NFIA, spermatocytes express NFIX, round spermatids express NFIB, and peritubular myoid cells express each of these three family members. Further analysis of NFIX expression during the cycle of the seminiferous epithelium revealed expression not in spermatogonia, as we anticipated, but in spermatocytes. These data suggested a potential role for NFIX in spermatogenesis. To investigate, we analyzed mice with constitutive deletion of Nfix (Nfix-null). Assessment of germ cells in the postnatal day 20 (P20) testes of Nfix-null mice revealed that spermatocytes initiate meiosis, but zygotene stage spermatocytes display structural defects in the synaptonemal complex, and increased instances of unrepaired DNA double-strand breaks. Many developing spermatocytes in the Nfix-null testis exhibited multinucleation. As a result of these defects, spermatogenesis is blocked at early diplotene and very few round spermatids are produced. Collectively, these novel data establish the global requirement for NFIX in correct meiotic progression during the first wave of spermatogenesis.

Summary Sentence

The transcription factor NFIX is required for meiotic progression during the first wave of spermatogenesis in the mouse.

Graphical Abstract

Glucose is a key substrate for supporting sperm energy production and function. Previous studies have demonstrated that sperm glucose uptake is facilitated by several isoforms of the glucose transporters (GLUT). Here, we report that sperm also expresses the Na⁺-dependent sodium glucose cotransporter (SGLT). This was first suggested by our observation that genetic deletion of the testis-specific Na,K-ATPase α4, which impairs the sperm plasma membrane Na⁺ gradient, reduces glucose uptake and ATP production. Immunoblot analysis revealed the presence of an SGLT in sperm, with specific expression of isoform 1 (SGLT-1), but not of isoform 2 (SGLT-2). Immunocytochemistry identified SGLT-1 in the mid- and principal piece of the sperm flagellum. Inhibition of SGLT-1 with the isotype-selective inhibitor phlorizin significantly reduced glucose uptake, glycolytic activity, and ATP production in noncapacitated and capacitated sperm from wild-type mice. Phlorizin also decreased total sperm motility, as well as other parameters of sperm movement. In contrast, inhibition of SGLT-1 had no significant effect on sperm hyperactivation, protein tyrosine phosphorylation, or acrosomal reaction. Importantly, phlorizin treatment impaired the fertilizing capacity of sperm. Altogether, these results demonstrate that mouse sperm express a functional SGLT transport system that is important for supporting sperm energy production, motility, and fertility.

Summary Sentence

The Na⁺-dependent glucose cotransporter type 1 (SGLT-1) is expressed in mouse spermatozoa and plays an important role in sperm physiology.

Graphical Abstract

Glutathione (GSH) is a tripeptide thiol antioxidant that has been shown to be important to overall reproductive health. Glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis consists of a catalytic and a modifier (GCLM) subunit. We previously showed that oxidative stress in the ovary and oocytes of Gclm^-/- mice is associated with accelerated age-related decline in ovarian follicles and decreased female fertility due to preimplantation embryonic mortality. Mammalian preimplantation development is a highly regulated and energy-intensive process that primarily relies on coordination between lipid droplets (LDs) and mitochondria to maintain cellular homeostasis. In this study, we hypothesized that GSH deficiency in oocytes increases oxidative stress, leading to increased mitochondrial dysfunction and decreased LD consumption, thereby decreasing oocyte developmental competence. We observed that Gclm^-/- oocytes have increased oxidative stress, primarily in the form of mitochondrial superoxide and decreased subcortical mitochondrial clusters. Further, Gclm^-/- oocytes have decreased mitochondrial membrane potential (ΔΨm) compared with Gclm^+/+. We surmise this is likely due to the decreased availability of LDs, as we observed a significant decrease in LD content in Gclm^-/- oocytes compared with Gclm^+/+. The decreased oocyte LD content is likely related to an altered serum lipidome, with Gclm^-/- serum having relatively lower unsaturated fatty acids and triglycerides than that of Gclm^+/+ and Gclm^+/- females. Altogether these data support that decreased LDs and increased oxidative stress are primary drivers of decreased oocyte developmental competence in GSH-deficient oocytes.

Summary Sentence

Glutathione deficiency leads to increased oocyte oxidative stress, reduced mitochondrial membrane potential, and aberrant lipid storage, reducing oocyte competence.

Graphical Abstract

The syncytial groups of germ cells (germ-line cysts) forming in ovaries of clitellate annelids are an attractive model to study mitochondrial stage-specific changes. Using transmission electron microscopy, serial block-face scanning electron microscopy, and fluorescent microscopy, we analyzed the mitochondria distribution and morphology and the state of membrane potential in female cysts in Enchytraeus albidus. We visualized in 3D at the ultrastructural level mitochondria in cysts at successive stages: 2-celled, 4-celled, 16-celled cysts, and cyst in advanced oogenesis. We found that mitochondria form extensive aggregates—they are fused and connected into large and branched mitochondrial networks. The most extensive networks are formed with up to 10 000 fused mitochondria, whereas individual organelles represent up to 2% of the total mitochondrial volume. We classify such a morphology of mitochondria as a dynamic hyperfusion state and suggest that this can maintain their high activity and intensify the process of cellular respiration within the syncytial cysts. We found some individual mitochondria undergoing degradation, which implies that damaged mitochondria are removed from networks for their final elimination. As growing oocyteswere shown to possess less active mitochondria than the nurse cells, the high activity of mitochondria in the nurse cells and their dynamic hyperfusion state are attributed to serve the needs of the growing oocyte. In addition, we measured by calorimetry the total antioxidant capacity of germ-line cysts in comparison with somatic tissue, and it suggests that antioxidative defense systems, together with mitochondrial networks, can effectively protect germ-line mitochondria from damage.

Summary Sentence

In the syncytial germ-line cysts of annelid Enchytraeus albidus, functioning during oogenesis, mitochondria form extensive networks that serve the needs of the growing oocyte.

Graphical Abstract

As a species without master sex-determining genes, zebrafish displays high plasticity in sex differentiation, making it an excellent model for studying the regulatory mechanisms underlying gonadal differentiation and gametogenesis. Despite being a gonochorist, zebrafish is a juvenile hermaphrodite that undergoes a special phase of juvenile ovary before further differentiation into functional testis and ovary. The mechanisms underlying juvenile ovary formation and subsequent gonadal differentiation remain largely unknown. In this study, we explored the role of Nobox/nobox (new born ovary homeobox protein), another oocyte-specific transcription factor in females, in early zebrafish gonadogenesis using CRISPR/Cas9 technology. As in mammals, nobox is specifically expressed in zebrafish gonads with a dimorphic pattern at juvenile stage. In contrast to the mutant of figla (factor in the germline alpha, another oocyte-specific transcription factor), the nobox mutants showed formation of typical perinucleolar (PN) follicles at primary growth (PG) stage in juvenile gonads, suggesting occurrence of follicle assembly from cystic oocytes (chromatin nucleolar stage, CN). These follicles, however, failed to develop further to form functional ovaries, resulting in all-male phenotype. Despite its expression in adult testis, the loss of nobox did not seem to affect testis development, spermatogenesis and male spawning. In summary, our results indicate an important role for Nobox in zebrafish ovarian differentiation and early folliculogenesis.

Graphical Abstract

Using genome-editing method, we demonstrated that Nobox, an oocyte-specific transcriptional factor, plays an essential role in ovarian formation during zebrafish gonadal differentiation and folliculogenesis in females. Although follicles could be seen to some extent in the absence of Nobox, their formation and further development are both retarded.

Placental insufficiency disorders are major obstetric complications that share a common phenomenon of poor placental trophoblast cell invasion and remodeling of uterine tissues. Myostatin is a transforming growth factor (TGF)-β superfamily member well known for its important role in muscle growth control. Myostatin is also produced in the placenta and has been shown to regulate some trophoblast functions. However, its roles in placental development are still poorly understood. In this study, we tested the hypothesis that myostatin increases trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling. Primary and immortalized (HTR8/SVneo) trophoblast cells were used as study models. Matrigel-coated transwell invasion assays were used to study the effects of recombinant human myostatin on trophoblast cell invasion. Reverse transcription quantitative real-time polymerase chain reaction and Western blot were used to measure myostatin effects on N-cadherin mRNA and protein levels, respectively. Small inhibitor molecules as well as siRNA-mediated knockdown were used to block myostatin receptor and downstream signaling, respectively. Data were analyzed either by unpaired Student T test or one-way analysis of variance followed by Newman Keuls test for multiple group comparisons. Myostatin significantly increased primary and HTR8/SVneo trophoblast cell invasion. Moreover, myostatin upregulated N-cadherin mRNA and protein levels in a time-dependent manner in both study models. These effects were blocked by inhibition of TGF-β type I receptors as well as siRNA-mediated knockdown of SMAD2/3 combined or common SMAD4. Importantly, myostatin-induced trophoblast cell invasion was abolished by knockdown of N-cadherin, SMAD2/3, or SMAD4. Myostatin may increase human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling.

Summary Sentence Myostatin increases primary and immortalized human trophoblast cell invasion and N-cadherin production. N-cadherin upregulation is required for myostatin-induced invasion and is mediated by SMAD2/3-SMAD4 signaling.

Graphical Abstract

Bisphenol A (BPA) exposure during pregnancy is associated with low fetal weight, particularly in male fetuses. The expression of estrogen-related receptor gamma (ESRRG), a receptor for BPA in the human placenta, is reduced in fetal growth restriction. This study sought to explore whether ESRRG signaling mediates BPA-induced placental dysfunction and determine whether changes in the ESRRG signaling pathway are sex-specific. Placental villous explants from 18 normal term pregnancies were cultured with a range of BPA concentrations (1 nM–1 µM). Baseline BPA concentrations in the placental tissue used for explant culture ranged from 0.04 to 5.1 nM (average 2.3 ±1.9 nM; n = 6). Expression of ESRRG signaling pathway constituents and cell turnover were quantified. BPA (1 µM) increased ESRRG mRNA expression after 24 h in both sexes. ESRRG mRNA and protein expression was increased in female placentas treated with 1 µM BPA for 24 h but was decreased in male placentas treated with 1 nM or 1 µM for 48 h. Levels of 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and placenta specific-1 (PLAC1), genes downstream of ESRRG, were also affected. HSD17B1 mRNA expression was increased in female placentas by 1 µM BPA; however, 1 nM BPA reduced HSD17B1 and PLAC1 expression in male placentas at 48 h. BPA treatment did not affect rates of proliferation, apoptosis, or syncytiotrophoblast differentiation in cultured villous explants. This study has demonstrated that BPA affects the ESRRG signaling pathway in a sex-specific manner in human placentas and a possible biological mechanism to explain the differential effects of BPA exposure on male and female fetuses observed in epidemiological studies.

Summary Sentence

BPA affects the ESRRG signaling pathway in a sex-specific manner in human placentas and is a possible biological mechanism to explain the differential effects of BPA exposure on male and female fetuses observed in epidemiological studies.

Fetal growth depends on placental function, which requires energy from mitochondria. Here we investigated whether mitochondrial function in the placenta relates to the growth of the lightest and heaviest fetuses of each sex within the litter of mice. Placentas from the lightest and heaviest fetuses were taken to evaluate placenta morphology (stereology), mitochondrial energetics (high-resolution respirometry), mitochondrial regulators, nutrient transporters, hormone handling, and signaling pathways (qPCR and Western blotting). We found that mitochondrial complex I and II oxygen consumption rate was greater for placentas supporting the lightest female fetuses, although placental complex I abundance of the lightest females and complexes III and V of the lightest males were decreased compared to their heaviest counterparts. Expression of mitochondrial biogenesis (Nrf1) and fission (Drp1 and Fis1) genes was lower in the placenta from the lightest females, whilst biogenesis-related gene Tfam was greater in the placenta of the lightest male fetuses. In addition, placental morphology and steroidogenic gene (Cyp17a1 and Cyp11a1) expression were aberrant for the lightest females, but glucose transporter (Slc2a1) expression was lower in only the lightest males versus their heaviest counterparts. Differences in intra-litter placental phenotype were related to changes in the expression of hormone-responsive (androgen receptor) and metabolic signaling (AMPK, AKT, and PPARγ) pathways. Thus, in normal mouse pregnancy, placental structure, function, and mitochondrial phenotype are differentially responsive to the growth of the female and male fetus. This study may inform the design of sex-specific therapies for placental insufficiency and fetal growth abnormalities with life-long benefits for the offspring.

Summary Sentence

Placenta structure, function, and mitochondrial functional capacity relate to the growth of the lightest and heaviest fetuses within the litter, and the nature of these changes differ in the two fetal sexes.

Graphical Abstract

Here, we showed that in normal mouse pregnancy, placenta function varies between the lightest and the heaviest female and male fetuses within the litter. In particular, there are differences in mitochondria function, structure, nutrient transporters, metabolic pathways, and steroid signaling in the placental transport zone (labyrinth zone) that relate to fetal growth in the litter.

Using mice with Y chromosome deficiencies and supplementing Zfy transgenes, we, and others, have previously shown that the loss of Y chromosome Zfy1 and Zfy2 genes is associated with infertility and spermiogenic defects and that the addition of Zfy transgenes rescues these defects. In these past studies, the absence of Zfy was linked to the loss of other Y chromosome genes, which might have contributed to spermiogenic phenotypes. Here, we used CRISPR/Cas9 to specifically remove open reading frame of Zfy1, Zfy2, or both Zfy1 and Zfy2, and generated Zfy knockout (KO) and double knockout (DKO) mice. Zfy1 KO and Zfy2 KO mice were both fertile, but the latter had decreased litters size and sperm number, and sperm headshape abnormalities. Zfy DKO males were infertile and displayed severe spermatogenesis defects. Postmeiotic arrest largely prevented production of sperm and the few sperm that were produced all displayed gross headshape abnormalities and structural defects within head and tail. Infertility of Zfy DKO mice could be overcome by injection of spermatids or sperm directly to oocytes, and the resulting male offspring had the same spermiogenic phenotype as their fathers. The study is the first describing detailed phenotypic characterization of mice with the complete Zfy gene loss. It provides evidence supporting that the presence of at least one Zfy homolog is essential for male fertility and development of normal sperm functional in unassisted fertilization. The data also show that while the loss of Zfy1 is benign, the loss of Zfy2 is mildly detrimental for spermatogenesis.

Summary Sentence

Mice with a complete loss of Y chromosome encoded Zfy1 and Zfy2 genes generated by CRISPR/Cas9-mediated open reading frame knockout are infertile and display severe spermatogenesis and sperm defects.