Microtubules are intracellular filaments that define in space and in time a large number of essential cellular functions such as cell division, morphology and motility, intracellular transport and flagella and cilia assembly. They are therefore essential for spermatozoon and oocyte maturation and function, and for embryo development. The dynamic and functional properties of the microtubules are in large part defined by various classes of interacting proteins including MAPs (microtubule associated proteins), microtubule-dependent motors, and severing and modifying enzymes. Multiple mechanisms regulate these interactions. One of them is defined by the high diversity of the microtubules themselves generated by the combination of different tubulin isotypes and by several tubulin post-translational modifications (PTMs). This generates a so-called tubulin code that finely regulates the specific set of proteins that associates with a given microtubule thereby defining the properties and functions of the network. Here we provide an in depth review of the current knowledge on the tubulin isotypes and PTMs in spermatozoa, oocytes, and preimplantation embryos in various model systems and in the human species. We focus on functional implications of the tubulin code for cytoskeletal function, particularly in the field of human reproduction and development, with special emphasis on gamete quality and infertility. Finally, we discuss some of the knowledge gaps and propose future research directions.

Summary Sentence

The tubulin code is emerging as an important regulator of microtubule function that impacts gamete and embryonic development.

Polycystic ovary syndrome (PCOS) is the most commonly diagnosed endocrine disorder in women of reproductive age, with phenotypes including ovarian and metabolic dysfunctions. Women with PCOS also show increased rates of mental illness, dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis, and altered responsiveness to stressors that may contribute to the higher rates of mental illness, specifically depression and anxiety.Animalmodels of PCOS have provided insight into the ovarian and metabolic mechanisms that underlie the syndrome, and several models have been used to study the behavioral consequences associated with PCOS in the laboratory. Several studies in rodent models of PCOS demonstrate changes in anxiety-like behavior, but researchers often neglect to report procedural details or behavioral data crucial to interpreting the differences observed in those studies. Additionally, the impact of potential HPA dysregulation in animalmodels of PCOS may influence behavioral findings, although only three studies to date have examined this. As such, researchers should consider and report stress-associated variables (e.g., time of day, light/dark cycle, light intensity, housing, and procedures to control experimenter and litter effects) that may influence depression- and anxiety-like behaviors in rodents. This review will summarize the behavioral and HPA-related studies in women with PCOS and rodent models of the disease, and provide considerations for future studies.

Summary Sentence

Women with PCOS have increased depression and anxiety, as well as HPA dysregulation, which should be taken into consideration when performing behavior studies in rodent models of PCOS.

Methionine adenosyltransferase II (MAT2A) is essential to the synthesis of S-adenosylmethionine, a major methyl donor, from L-methionine and ATP. Upon fertilization, zygotic genome activation (ZGA) marks the period that transforms the genome from transcriptional quiescence to robust transcriptional activity. During this period, embryonic epigenome undergoes extensive modifications, including histone methylation changes. However, whether MAT2A participates in histone methylation at the ZGA stage is unknown. Herein, we identified that MAT2A is a pivotal factor for ZGA in mouse embryos. Mat2a knockdown exhibited 2-cell embryo arrest and reduced transcriptional activity but did not affect H3K4me2/3 and H3K9me2/3. When the cycloleucine, a selective inhibitor of MAT2A catalytic activity, was added to a culture medium, embryos were arrested at the morula stage in the same manner as the embryos cultured in an L-methionine-deficient medium. Under these two culture conditions, H3K4me3 levels of morula and blastocyst were much lower than those cultured under normal medium. Furthermore, cycloleucine treatment or methionine starvation apparently reduced the developmental potential of blastocysts. Thus, Mat2a is indispensable for ZGA and morula-to-blastocyst transition.

Summary Sentence

Mat2a is pivotal for transcriptional activation of mouse 2-cell embryos and transition from morula to blastocyst

To date, very few studies have reported on the relationship between live birth gender and embryo development kinetics. This study included 1735 women undergoing in vitro fertilization or intracytoplasmic sperm injection by using a time-lapse system. Finally, a total of 228 qualified patients with 100% implantation and known live birth information were included in the analysis. There were 174 male live births and 134 female live births. The time to 3 (t3), 4 (t4), and 5 (t5) cell development of male embryos was significantly shorter/earlier than female embryos (P < 0.05). The duration of the second cell cycle (cc2) in male embryos was significantly shorter than female embryos (P = 0.002). Multivariate logistic regression showed that only t3 had a significant correlation with live birth gender; the odds ratio (OR) was 0.786, 95% confidence interval (CI) was 0.625–0.988 (P < 0.05). When morphokinetic parameters were divided into groups based on quartiles, embryos within the sex ranges were observed to have significantly different proportions of male and female live births (P < 0.05). The results showed that t3 (<14 h) was the most relevant parameter related to live birth gender (OR 2.452, 95% CI 1.071–5.612, P = 0.03). These findings support the idea that embryo morphokinetic parameters were affected by the sex of the embryo. Currently, embryologists use embryo morphokinetics to establish models of development, in order to improve accurate selection of viable embryos. Thus, this factor needs to be considered when embryologists use embryo morphokinetics to select embryos.

Summary Sentence

Embryo morphokinetic parameters were affected by the sex of the embryo. This factor needs to be considered when embryologists use embryo morphokinetics to select embryos.

Maternal exposure to estrogens can induce long-term adverse effects in the offspring. The epigenetic programming may start as early as the period of preimplantation development. We analyzed the effects of gestational estradiol-17β (E2) exposure with two distinct low doses, corresponding to the acceptable daily intake “ADI” and close to the no-observed-effect level “NOEL”, and a high dose (0.05, 10, and 1000 µg E2/kg body weight daily, respectively). The E2 doses were orally applied to sows from insemination until sampling at day 10 of pregnancy and compared to carrier-treated controls leading to a significant increase in E2 in plasma, bile and selected somatic tissues including the endometrium in the high-dose group. Conjugated and unconjugated E2 metabolites were as well elevated in the NOEL group. Although RNA-sequencing revealed a dose-dependent effect of 14, 17, and 27 differentially expressed genes (DEG) in the endometrium, single embryos were much more affected with 982 DEG in female blastocysts of the high-dose group, while none were present in the corresponding male embryos. Moreover, the NOEL treatment caused 62 and 3 DEG in female andmale embryos, respectively. Thus, we detected a perturbed sex-specific gene expression profile leading to a leveling of the transcriptome profiles of female and male embryos. The preimplantation period therefore demonstrates a vulnerable time window for estrogen exposure, potentially constituting the cause for lasting consequences. The molecular fingerprint of low-dose estrogen exposure on developing embryos warrants a careful revisit of effect level thresholds.

Summary Sentence

Maternal oral low-dose estrogen exposure during the preimplantation period specifically targeted female embryos by inducing a male-like gene expression profile.

Polycystic ovary syndrome (PCOS) is an endocrine disorder in women of reproductive age. In addition to anovulation, endometrial dysfunction can reduce fertility in PCOS. The cyclical changes of endometrium are controlled by estrogen and progesterone via modulating the Wnt/B-catenin pathway. Clomiphene citrate (CC) and letrozole are used to induce ovulation; unlike letrozole, there is a discrepancy between ovulation and pregnancy rates in CC-treated cycles. Because of the antiestrogenic effects of CC on endometrium, we compared the expression of the key molecules of the Wnt/B-catenin pathway in the endometrium of women taking CC and letrozole. This study included PCOS and healthy women divided into the groups stimulated with letrozole (5 mg) or CC (100 mg) as well as NO-treatment groups. The endometrial thickness and hormonal profile were measured on day 12 of the menses. Using real-time polymerase chain reaction and western blot, we evaluated mRNA and protein expression of B-catenin, glycogen synthase kinase 3 beta (GSK3B), dickkopf Wnt signaling pathway inhibitor 1 (DKK1), and estrogen receptor 1 (ESR1) in the endometrial samples. Significantly, the mean serum estrogen and progesterone were lower and higher, respectively, in letrozole than CC groups. The endometrial thickness was significantly reduced in CC. The proteins expression of active B-catenin, inactive GSK3B, and ESR1 were significantly decreased in CC-treated groups. The mRNA and protein assessment of DKK1 showed significantly higher expression in CC. Our results indicate that letrozole can provide an acceptable activation of the Wnt/B-catenin pathway, resulting in adequate proliferation of endometrium in the women receiving letrozole compared to CC.

Summary Sentence

The expression of B-catenin, GSK3B, DKK1, and ESR1 were adversely affected in the endometrium of women induced with clomiphene citrate compared to letrozole, resulting in inefficacity of endometrium.

Endometriosis is a common multifactorial gynecological disorder defined as the proliferation of endometrial tissue outside of the uterine cavity. Neuroangiogenesis (co-recruitment of nerves and blood vessels) is believed to play an integral part in the establishment and growth of endometriotic lesions. We hypothesized that exosomes derived from abnormal endometrium may serve as the second identifier of endometriosis and play an important role in the development of endometriosis by regulating neuroangiogenesis. Primary human endometrial stromal cells (ESCs) were isolated from eutopic endometrium (EmESC, n = 22) with endometriosis and normal endometrium (CoESC, n = 6). Exosomes were isolated from ESCs using the “standard” ultracentrifugation method, and the characterization of exosomes was identified through transmission electron microscopy, nanoparticle tracking analysis, and western blot. The role of exosomes in regulating neuroangiogenesis was determined through in vitro tube formation assay, neurite outgrowth assay, and dorsal root ganglion (DRG) neuron apoptosis analysis. The data showed that EmESCs could secrete exosomes with a diameter of approximately 100 nm and a biconcave morphological feature; they were internalized by human umbilical vein endothelial cells and DRG neurons and enhanced neuroangiogenic effects. We further validated the role of exosomes through blocking experiments.We found that when the exosome secretion was blocked, the pro-neuroangiogenesis effects were decreased. In conclusion, these data suggested that exosomes may play a key role in endometriosis by promoting neuroangiogenesis.

Summary Sentence

Exosomes may play a key role in endometriosis by promoting neuroangiogenesis.

Seminal plasma has conventionally been viewed as a transport and survival medium for mammalian sperm; however, its role now extends beyond this process to actively targeting female tissues. Studies in rodents, swine, and humans demonstrate that seminal plasma induces molecular and cellular changes within the endometrium or cervix following insemination. Seminal-plasmainduced alterations to the maternal environment have been theorized to facilitate embryo development, modulate maternal immunity toward the conceptus, and potentially improve pregnancy success. It is unknown if bovine seminal plasma modulates the uterine environment following insemination in the cow, where routine use of artificial insemination reduces maternal exposure to seminal plasma. We hypothesize that seminal plasma modulates the expression of inflammatory mediators in the endometrium, altering the maternal environment of early pregnancy. In vitro, seminal plasma altered intact endometrial explant expression of CSF2, IL1B, IL6, IL17A, TGFB1, IFNE, PTGS2, and AKR1C4. Furthermore, endometrial epithelial cell CSF2, CXCL8, TGFB1, PTGS2, and AKR1C4 expression were increased after seminal plasma exposure, while endometrial stromal cell CSF2, IL1B, IL6, CXCL8, IL17A, TGFB1, PTGS2, and AKR1C4 expression were increased following seminal plasma exposure. Endometrial expression of IL1B was increased in the cow 24 h after uterine infusion of seminal plasma, while other evaluated inflammatory mediators remained unchanged. These data indicate that seminal plasma may induce changes in the bovine endometrium in a temporal manner. Understanding the role of seminal plasma in modulating the maternal environment may aid in improving pregnancy success in cattle.

Summary Sentence

Exposure of endometrial cells to seminal plasma alters gene expression of inflammatory mediators, reminiscent of the postcoital inflammatory response in other species.

Pregnancy establishment in cattle is contingent on conceptus elongation—a fundamental developmental event coinciding with the time during which most pregnancies fail. Elongation in vivo is directly driven by uterine secretions, indirectly influenced by systemic progesterone concentrations, and has yet to be recapitulated in vitro. To better understand the microenvironment evolved to facilitate this phenomenon, the amino acid and carbohydrate composition of uterine fluid was interrogated using high-throughput metabolomics on days 12, 13, and 14 of the estrous cycle from heifers with normal and high circulating progesterone. A total of 99 biochemicals (79 amino acids and 20 carbohydrates) were consistently identified, of which 31 showed a day by progesterone interaction. Fructose and mannitol/sorbitol did not exhibit a day by progesterone interaction, but displayed the greatest individual fluctuations (P ≤ 0.05) with respective fold increases of 18.39 and 28.53 in high vs normal progesterone heifers on day 12, and increases by 10.70-fold and 14.85-fold in the uterine fluid of normal progesterone animals on day 14 vs day 12. Moreover, enrichment analyses revealed that the phenylalanine, glutathione, polyamine, and arginine metabolic pathways were among the most affected by day and progesterone. In conclusion, progesterone had a largely stabilizing effect on amino acid flux, and identified biochemicals of likely importance to conceptus elongation initiation include arginine, fructose, glutamate, and mannitol/sorbitol.

Summary Sentence

Day 3 progesterone supplementation alters the amino acid and carbohydrate profile of the bovine uterine fluid on days 12, 13, and 14 postestrus—the window coinciding with conception elongation initiation.

The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct.

Summary Sentence

ADAM6, a testis-specific protein, is required for sperm ascent into the oviduct and its deletion results in deficits in both sperm–sperm and sperm–extracellular matrix associations.

The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XY^sry− mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XY^sry− females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XY^sry− PGCs was associated with aberrant β-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XY^sry− oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as “Swyer syndrome,” in which patients with an XY karyotype present as typical females and are infertile.

Summary Sentence

We report the molecular etiology of XY oocyte dysfunction in sex-reversed female mice harboring Sry deletion. The results show potential molecular causes for germ cell disruption providing insights into disorders of sex differentiation in humans.

Actin filaments are widely involved in multiple cellular processes in oocytemeiosis, such as spindle migration and polar body extrusion. The actin nucleators like Arp2/3 complex and formins are the most recognized molecules for actin assembly in oocytes. In the present study, we report that the vesicle trafficking factor, RAB8A GTPase, is a new regulator critical for actin assembly in meiosis. Our results showed that RAB8A was localized at both the spindle periphery and cortex in mouse oocytes, which was similar to the localization patterns of actin filaments. RAB8A depletion caused spindle migration defects and the failure of polar body extrusion, which could have been due to decreases in both cytoplasmic and cortical actin filaments in oocytes. Based on mass spectrometry analysis, we showed that RAB8A promoted actin assembly through its modulation on the ROCK-LIMK signaling pathway. Moreover, we demonstrated that RAB8A colocalized and interacted with GM130 at the spindle periphery and that RAB8A depletion caused the disruption of GM130-docked Golgi distribution. Taken together, our data indicated that RAB8A was required for Golgi distribution, spindlemigration, and polar body extrusion via ROCK-mediated actin assembly in mouse oocyte meiosis.

Summary Sentence

The small GTPase RAB8A regulates ROCK for actin assembly, which further affects the meiotic spindle migration and polar body extrusion in mouse oocyte meiosis. We also found that RAB8A interacts with GM130 for Golgi apparatus distribution.

Four isoforms of serine/threonine phosphatase type I, PP1α, PP1β, PP1γ 1, and PP1γ 2, are derived from three genes. The PP1γ 1 and PP1γ 2 isoforms are alternately spliced transcripts of the protein phosphatase 1 catalytic subunit gamma gene (Ppp1cc). While PP1γ 1 is ubiquitous in somatic cells, PP1γ 2 is expressed exclusively in testicular germ cells and sperm. Ppp1cc knockout male mice (–/–), lacking both PP1γ 1 and PP1γ 2, are sterile due to impaired sperm morphogenesis. Fertility and normal sperm function can be restored by transgenic expression of PP1γ 2 alone in testis of Ppp1cc (–/–) mice. The purpose of this study was to determine whether the PP1γ 1 isoform is functionally equivalent to PP1γ 2 in supporting spermatogenesis and male fertility. Significant levels of transgenic PP1γ 1 expression occurred only when the transgene lacked a 1-kb 3′UTR region immediately following the stop codon of the PP1γ 1 transcript. PP1γ 1 was also incorporated into sperm at levels comparable to PP1γ 2 in sperm from wild-type mice. Spermatogenesis was restored in mice expressing PP1γ 1 in the absence of PP1γ 2. However, males from the transgenic rescue lines were subfertile. Sperm from the PP1γ 1 rescue mice were unable to fertilize eggs in vitro. Intrasperm localization of PP1γ 1 and the association of the protein regulators of the phosphatase were altered in epididymal sperm in transgenic PP1γ 1 compared to PP1γ 2. Thus, the ubiquitous isoform PP1γ 1, not normally expressed in differentiating germ cells, could replace PP1γ 2 to support spermatogenesis and spermiation. However, PP1γ 2, which is the PP1 isoform in mammalian sperm, has an isoform-specific role in supporting normal sperm function and fertility.

Summary Sentence

PP1γ 1 can replace PP1γ2 in testis.

Neonatal exposure to high-dose 17β-estradiol (E2) affects the morphology and physiology of sex and accessory sex organs in the long term. In this study, we examined the effects of E2 imprinting on male sexual behavior, fertility, and the number of androgen receptor (AR)-expressing cells in the hypothalamus. E2-treated males showed copulatory behavior represented by mounts and/or intromissions, demonstrating the preservation of aspects of male behavior. They had slightly increased latency for first intromission and a reduced number of ejaculations, associated with a 50% reduction in the fertility index. AR expression in the hypothalamus was assessed by RT-PCR, western blotting, and immunohistochemistry. Treated rats had a significantly lower ventral prostate (VP) weight, demonstrating the efficacy of the treatment. The AR mRNA and protein content in the hypothalamus of E2-treated animals was reduced to the levels of females. AR-expressing cell counts in the ventromedial, anterior medial preoptic, paraventricular nuclei, and preoptic areas were different from control males, and similar to those of females. In conclusion, E2 imprinting resulted not only in ill-developed sexual organs, but also affected sexual behavior, resulting in a female-type hypothalamus, at least with respect to the abundance of AR mRNA and protein and the number of AR-expressing cells in important regions/tracts.

Summary Sentence

Neonatal exposure to high-dose estradiol affects the number of AR+ hypothalamic neurons and male sexual behavior.

Estrogen plays a pivotal role in the sex differentiation of teleosts, whereas the precise function of androgens is more controversial. In this study, orange-spotted grouper (Epinephelus coioides) fry were treated with letrozole (an aromatase inhibitor, AI), 17α-methyltestosterone (MT), or MT and 17β-estradiol (E2) simultaneously, during the period of gonadal formation and sex differentiation. MT feeding at 50 days after hatching resulted in gonadal dysgenesis, which could be rescued by E2 supplementation. Different doses of AI treatment led to different phenotypes: undifferentiated gonads weremaintained in the AI group fed a low dose (5mg/kg diet), whereas female-to-male sex reversal was observed in the AI group fed a high dose (100 mg/kg diet). MT andMT + E2 treatment could induce female-to-male sex reversal during sex differentiation (90 days after hatching). The expression of female pathway genes was suppressed, while the expression of genes in the male pathway was up-regulated in the MT + E2 group. Consistent with the expression of sex-related genes, the serum 11- ketotestosterone level was also upregulated in MT and MT + E2 group. Finally, we examined the expression of male-specific mark (DMRT1) and proliferating cell nuclear antigen in MT and MT + E2 induced sex reversal, and the result indicated that male germ cells and somatic cells may origin from the gonium and proliferative somatic cells surrounding the efferent duct, respectively. Overall, our data suggested that estrogen acts as a natural inducer of female differentiation, and that the co-administration of estrogen and androgen during sex differentiation leads to a male sex fate in the protogynous orange-spotted grouper.

Summary Sentence

We systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels and cellular changes during sex changes induced by MT+E2 during sex differentiation in the protogynous orange-spotted grouper.

Estrogens are involved in a wide range of processes in vertebrate reproduction through ligand activation of their specific cognate receptors. In most teleosts, three nuclear estrogen receptor subtypes have been identified (Esr1, Esr2a, and Esr2b). Differences in ligand binding affinity and seasonal expression patterns in reproductive tissues among these Esr subtypes suggest distinct roles during oogenesis, vitellogenesis, and spermatogenesis. This study focuses on the role of the Esr subtypes in European sea bass (Dicentrarchus labrax) oogenesis and their endocrine regulation. The coding genes of the three Esr subtypes are highly expressed in reproduction-related tissues such as pituitary, gonad, and liver. Quantification of esr1, esr2a, and esr2b expression in the ovary and liver during a whole reproductive cycle showed different patterns depending on stage and subtype, suggesting differential roles of the three receptors in the regulation of oogenesis and vitellogenesis. Esr2a and Esr2b also showed differences in transcriptional activity and ligand affinity when functionally characterized in HEK293 cells. Finally, for the first time in teleosts, the localization of the three Esr subtypes in ovarian follicles and their regulation by gonadotropins is described. Immunodetection of the receptors revealed different distribution patterns in follicular cells and various subcellular locations of the oocyte. Gonadotropin stimulation of ovarian follicles in different stages of vitellogenesis showed a consistent induction of esrb2b expression by Fsh. All together, these data reinforce the hypothesis that each estrogen receptor plays a specific role in oogenesis.

Summary Sentence

The three sea bass Esr subtypes have different ligand affinities and tissue expression patterns. In liver, only sbEsr1 and sbEsr2b play a role in estradiol-17β induced vitellogenesis. In ovary, sbEsr2b appears to be involved in vitellogenesis, while sbEsr1 and sbEsr2a seem to be implicated in maturation. All are localized in somatic and germ cells. Follicle-stimulating hormone regulates sbesr2b but not sbesr1 and sbesr2a transcription.

Preterm prelabor rupture of membranes (PPROM), which can precede or follow intra-amniotic infection/inflammation (IAI), is a poorly understood pregnancy complication. Tenascin-X (TNX) is a connective tissue extracellular matrix protein that regulates fibrillogenesis of collagens I, III, and V. Our goal was to investigate the presence and level of soluble TNX (sTNX) in amniotic fluid (AF) and TNX expression in reproductive tissues of pregnancies complicated by PPROM and IAI. We prospectively recruited 334 women pregnant with singletons who had a clinically indicated amniocentesis for genetic karyotyping, lung maturity testing, or rule-out IAI in the presence or absence of PPROM. We quantified TNX expression in fetal membranes, myometrium, cervix, and placenta using immunological methods and qRT-PCR. In pregnancies with normal outcomes, AF sTNX levels were GA-regulated with lower levels toward term. IAI significantly upregulated AF sTNX levels independent of membrane status. AF sTNX levels inversely correlated with fetalmembranes tenascin XB (TNXB) mRNA level, which was significantly downregulated by IAI. Western blotting identified characteristic ∼⃒75 and ∼⃒140 kDa sTNX forms in both AF and fetal membranes. Fetal membranes, placenta, and cervix constitutively express TNX with the highest abundance in the amnion. Amnion TNX richness is significantly lost in the setting of IAI. Our results suggest that fetal membranes may be a source of AF sTNX whereby protein and mRNA expression seem to be significantly impacted by inflammation independent of fetal membrane status. A more thorough understanding of TNX changes may be valuable for understanding spontaneous PPROM and to potentially develop therapeutic targets.

Summary Sentence

Fetal membranes may be a source of amniotic fluid soluble tenascin-X, with both protein and mRNA levels significantly affected by inflammation independent of fetal membrane status.

Birth is a complex biological event requiring genetic, cellular, and physiological changes to the uterus, resulting in a uterus activated for completing the physiological processes of labor. We define the change from the state of pregnancy to the state of parturition as uterine transitioning, which requires the actions of inflammatorymediators and localized paracrine interactions between intrauterine tissues. Few studies have examined the in vitro interactions between fetal and maternal gestational tissues within this proinflammatory environment. Thus, we designed a co-culture model to address this gap, incorporating primary term human myometrium smooth muscle cells (HMSMCs) with human fetal membrane (hFM) explants to study interactions between the tissues. We hypothesized that crosstalk between tissues at term promotes proinflammatory expression and uterine transitioning for parturition. Outputs of 40 cytokines and chemokines encompassing a variety of proinflammatory roles were measured; all but one increased significantly with coculture. Eighteen of the 39 cytokines increased to a higher abundance than the sum of the effect of each tissue cultured separately. In addition, COX2 and IL6 but not FP and OXTR mRNA abundance significantly increased in both HMSMCs and hFM in response to co-culture. These data suggest that synergistic proinflammatory upregulation within intrauterine tissues is involved with uterine transitioning.

Summary Sentence

Intrauterine tissue crosstalk, measured in vitro by co-culture of human fetal membrane tissue explants with primary human myometrium smooth muscle cells, promotes proinflammatory amplification and uterine transition for labor.

The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphroditic grouper (Epinephelus fuscoguttatus) was investigated. Recombinant giant grouper (E. lanceolatus) FSH (rggFSH) was produced in yeast. Its receptor-binding capacity and steroidogenic potency were confirmed in vitro. Weekly injections of rggFSH to juvenile tiger grouper for 8 weeks (100 µg/kg body weight, BW) resulted in significantly larger and more advanced oocytes (cortical alveolar stage vs primary growth stage in control). Sustained treatment with rggFSH (20 to 38 weeks at 200 µg/kg BW) resulted in significant reduction in gonad size, degeneration of oocytes, and proliferation of spermatogonial cells, indicative of female to male sex change. Gene expression analysis showed that, while initiating female to male sex change, the rggFSH significantly suppressed the steroidogenic genes cyp11b, cyp19a1a, and foxl2 which restrained the endogenous production of sex steroid hormones and thus prevented the differentiation of spermatogonial cells. Expression profile of sex markers dmrt1, amh, figla, and bmp15 suggests that the observed sex change was restricted at the initiation stage. Based on these results, we propose that the process of female to male sex change in the protogynous grouper is initiated by FSH, rather than sex steroids, and likely involves steroid-independent pathway. The cortical alveolar stage in oocyte development is the critical point after which FSH-induced sex change is possible in grouper.

Summary Sentence

Administration of recombinant grouper FSH to juvenile protogynous grouper promoted oocyte development to the cortical alveolar stage, and initiated female tomale sex change while suppressing steroidogenic activity.

We previously demonstrated that in the mouse only two Y chromosome genes are required for a male to produce an offspring with the help of assisted reproduction technologies (ART): testis determinant Sry and spermatogonial proliferation factor Eif2s3y. Subsequently, we have shown that the function of these genes can be replaced by transgenic overexpression of their homologs, autosomally encoded Sox9 and X-chromosome encoded Eif2s3x. Males with Y chromosome contribution limited to two (X^Eif2s3yOSry), one (X^Eif2s3yOSox9 and XOSry,Eif2s3x), and no genes (XOSox9,Eif2s3x) produced haploid germ cells and sired offspring after ART. However, despite successful assisted reproductive outcome, they had smaller testes and displayed abnormal development of the seminiferous epithelium and testicular interstitium. Here we explored whether these testicular defects originated from altered pro-testis and pro-ovary factor signaling in genital ridges at the time of sex determination. Timed pregnancies were generated to obtain transgenic X^Eif2s3yOSry, X^Eif2s3yOSox9, XOSry,Eif2s3x, XOSox9,Eif2s3x, and wild-type XX and XY fetuses at 12.5 days post coitum. Dissected genital ridges were assessed for their morphology and anatomy, and expression of pro-testis and pro-ovary transcripts. All transgenic males displayed incomplete masculinization of gonadal shape, impaired development of testicular cords and gonadal vasculature, and decreased expression of factors promoting male pathway. Fetal gonadmasculinization was more effective when sex determination was driven by the Sry transgene, in the presence of Y chromosome genes, and to a lesser extent a double dosage of X genes. The study adds to the understanding of the role of Y chromosome genes and their homologs during sex determination.

Summary Sentence

Males with limited Y chromosome gene contribution undergo incomplete gonadalmasculinization during fetal development.

We reported that FGIN-1-27 (N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide, FGIN), a synthetic ligand for translocator protein (TSPO, 18 kDa), increased serum testosterone levels in young and aged Brown Norway rats after its administration daily for 10 days. It is not known, however, how soon after treatment with FGIN serum testosterone rises, how long levels remain elevated after cessation of treatment, or whether the drug acts solely through TSPO. Adult Sprague-Dawley male rats received a single ip dose of FGIN (1mg/kg BW). Serial blood samples were collected, and serum testosterone and luteinizing hormone (LH) were assessed hourly throughout 24 h. Testosterone concentration was maximal by 3 h, remained significantly higher than the controls at 10 h, and returned to the control level by 24 h. Consistent with the in vivo study, culturing isolated Leydig cells with either FGIN (40 µM) or LH (0.1 ng/ml) resulted in significantly increased testosterone production by 30 min, and the stimulatory effects persisted through 48 h. At a very early (15 min) treatment time, however, FGIN significantly increased testosterone production but LH had not yet done so. Surprisingly, in vivo treatment with FGIN not only increased serum testosterone but also serum LH concentration, raising the possibility that FGIN may increase serum testosterone concentration by dual mechanisms.

Summary Sentence

FGIN-1-27, a TSPO drug ligand, can acutely increase serum testosterone concentration by acting directly on the Leydig cells and indirectly through luteinizing hormone.

The progression of spermatogenesis is precisely controlled by meiotic stage-specific genes, but the molecular mechanism for activation of such genes is still elusive. Here we found a novel testis-specific long noncoding RNA (lncRNA), Tesra, that was specifically expressed in the mouse testis at the Prss/Tessp gene cluster on chromosome 9. Tesra was transcribed downstream of Prss44/Tessp-4, starting within the gene, as a 4435-nucleotide transcript and developmentally activated at a stage similar to that for Prss/Tessp genes. By in situ hybridization, Tesra was found to be localized in and around germ cells and Leydig cells, being consistent with biochemical data showing its existence in cytoplasmic, nuclear, and extracellular fractions. Based on the finding of more signals in nuclei of pachytene spermatocytes, we explored the possibility that Tesra plays a role in transcriptional activation of Prss/Tessp genes. By a ChIRP assay, the Tesra transcript was found to bind to the Prss42/Tessp-2 promoter region in testicular germ cells, and transient overexpression of Tesra significantly activated endogenous Prss42/Tessp-2 expression and increased Prss42/Tessp-2 promoter activity in a reporter construct. These findings suggest that Tesra activates the Prss42/Tessp-2 gene by binding to the promoter. Finally, we investigated whether Tesra co-functioned with enhancers adjacent to another lncRNA, lncRNA-HSVIII. In the Tet-on system, Tesra transcription significantly increased activity of one enhancer, but Tesra and the enhancer were not interdependent. Collectively, our results proposed a potential function of an lncRNA, Tesra, in transcriptional activation and suggest a novel relationship between an lncRNA and an enhancer.

Summary Sentence

A novel long noncoding RNA, Tesra, that is specifically expressed in the mouse testis activates a spermatocyte-specific gene, Prss42/Tessp-2, in cooperation with an enhancer by binding to chromatin at the promoter and enhancing its activity.

Systemic inflammation may impair male fertility, and its underlying mechanisms remain poorly understood. The present study investigates the effect of lipopolysaccharide (LPS)-induced systemic inflammation on the testis and epididymis in mice. Intraperitoneal injection of LPS significantly impaired testicular functions, including testosterone production, spermatogenesis, and blood–testis barrier permeability. The epididymitis characterized by leukocyte infiltration and fibrosis was observed in the cauda epididymis after LPS injection. LPS-induced testicular dysfunction and epididymitis were abolished in tumor necrosis factor alpha (Tnfa) knockout mice. Pomalidomide, a TNFA inhibitor, blocked the detrimental effects of LPS on the testis and epididymis. The results indicate that LPS-induced systemic inflammation impairs male fertility through TNFA production, suggesting that the intervention on TNFA production would be considered for the prevention and treatment of inflammatory impairment of male fertility.

Summary Sentence

LPS-induced systemic inflammation leads to testicular dysfunction and epididymitis through TNFA production in mice.