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Anthony D Horlock, Thomas J R Ormsby, Martin J D Clift, José E P Santos, John J Bromfield, and I Martin Sheldon

In brief

Bovine granulosa cells need to be cultured with serum to generate inflammation in response to bacterial lipopolysaccharide. This study shows that it is cholesterol that facilitates this lipopolysaccharide-stimulated cytokine secretion.

Abstract

During bacterial infections of the bovine uterus or mammary gland, ovarian granulosa cells mount inflammatory responses to lipopolysaccharide (LPS). In vitro, LPS stimulates granulosa cell secretion of the cytokines IL-1α and IL-1β and the chemokine IL-8. These LPS-stimulated inflammatory responses depend on culturing granulosa cells with serum, but the mechanism is unclear. Here, we tested the hypothesis that cholesterol supports inflammatory responses to LPS in bovine granulosa cells. We used granulosa cells isolated from 4 to 8 mm and >8.5 mm diameter ovarian follicles and manipulated the availability of cholesterol. We found that serum or follicular fluid containing cholesterol increased LPS-stimulated secretion of IL-1α and IL-1β from granulosa cells. Conversely, depleting cholesterol using methyl-β-cyclodextrin diminished LPS-stimulated secretion of IL-1α, IL-1β and IL-8 from granulosa cells cultured in serum. Follicular fluid contained more high-density lipoprotein cholesterol than low-density lipoprotein cholesterol, and granulosa cells expressed the receptor for high-density lipoprotein, scavenger receptor class B member 1 (SCARB1). Furthermore, culturing granulosa cells with high-density lipoprotein cholesterol, but not low-density lipoprotein or very low-density lipoprotein cholesterol, increased LPS-stimulated inflammation in granulosa cells. Cholesterol biosynthesis also played a role in granulosa cell inflammation because RNAi of mevalonate pathway enzymes inhibited LPS-stimulated inflammation. Finally, treatment with follicle-stimulating hormone, but not luteinising hormone, increased LPS-stimulated granulosa cell inflammation, and follicle-stimulating hormone increased SCARB1 protein. However, changes in inflammation were not associated with changes in oestradiol or progesterone secretion. Taken together, these findings imply that cholesterol supports inflammatory responses to LPS in granulosa cells.

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Alexis Gonzalez, Malia D Berg, Bruce Southey, and Matthew Dean

In brief

Glucose is an important nutrient for the endometrium and embryo during pregnancy. This study shows that estradiol (E2)/IGF1 signaling stimulates glycogen synthesis in the uterine epithelium of cows, which could provide glucose when needed.

Abstract

Glycogen storage in the uterine epithelium peaks near estrus and is a potential source of glucose for the endometrium and embryos. However, the hormonal regulation of glycogen synthesis in the uterine epithelium is poorly understood. Our objective was to evaluate the effect of E2 and insulin-like growth factor 1 (IGF1) on glycogenesis in immortalized bovine uterine epithelial (BUTE) cells. Treatment of BUTE cells with E2 (0.1–10 nM) did not increase glycogen levels. However, treatment of BUTE cells with IGF1 (50 or 100 ng/mL) resulted in a >2-fold increase in glycogen. To determine if the uterine stroma produced IGF1 in response to E2, bovine uterine fibroblasts were treated with E2, which increased IGF1 levels. Immunohistochemistry showed higher levels of IGF1 in the stroma on day 1 than on day 11, which coincides with higher glycogen levels in the uterine epithelium. Western blots revealed that IGF1 treatment increased the levels of phospho-AKT, phospho-GSKβ, hexokinase 1, and glycogen synthase in BUTE cells. Metabolomic (GC-MS) analysis showed that IGF1 increased 3-phosphoglycerate and lactate, potentially indicative of increased flux through glycolysis. We also found higher levels of N-acetyl-glucosamine and protein glycosylation after IGF1 treatment, indicating increased hexosamine biosynthetic pathway activity. In conclusion, IGF1 is produced by uterine fibroblasts due to E2, and IGF1 increases glucose metabolism and glycogenesis in uterine epithelial cells. Glycogen stored in the uterine epithelium due to E2/IGF1 signaling at estrus could provide glucose to the endometrium or be secreted into the uterine lumen as a component of histotroph.

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Jiarong Feng, Yanan Zhang, Xiaojian Yang, and Yan Zhang

In brief

The genetic heterogeneity of CFTR gene mutations in Chinese patients with congenital absence of the vas deferens (CAVD) differs from the hotspot mutation pattern in Caucasians. This paper reviews and suggests a more suitable screening strategy for the Chinese considering the dilemma of CFTR genetic blocking.

Abstract

Congenital absence of the vas deferens (CAVD) is a major cause of obstructive azoospermia and male infertility, with CFTR gene mutation as the main pathogenesis. Other genes such as ADGRG2, SLC9A3, and PANK2 have been discovered and proven to be associated with CAVD in recent studies. Multiple CFTR hotspot mutations have been found in Caucasians in several foreign countries, and relevant genetic counseling and preimplantation genetic diagnosis (PGD) have been conducted for decades. However, when we examined research on Chinese CAVD, we discovered that CFTR mutations show heterogeneity in the Chinese Han population, and there is currently no well-established screening strategy. Therefore, we have reviewed the literature, combining domestic and international research as well as our own, aiming to review research progress on the CFTR gene in China and discuss the appropriate scope for CFTR gene detection, the detection efficiency of other CAVD-related genes, and the screening strategy applicable to the Chinese Han population. This study provides more valuable information for genetic counseling and a theoretical basis for PGD and treatment for couples with CAVD when seeking reproductive assistance.

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Monika Fluks, Szymon Tamborski, Maciej Szkulmowski, and Anna Ajduk

In brief

Optical coherence microscopy is a label-free and non-invasive imaging technique capable of 3D subcellular structure visualization. Here we show that this method allows for quality assessment of immature mouse oocytes based on their chromatin conformation and can be a valuable addition to the toolkit used in assisted reproduction procedures.

Abstract

The success of assisted reproductive technologies, and particularly in vitro maturation, is tightly linked to the quality of oocytes. Therefore, there is a need for robust, reliable, and easy-to-assess biomarkers of oocyte developmental competence. Microscopy techniques visualizing oocyte intracellular structure could provide such biomarkers. However, fluorescence imaging methods, applied frequently in biology and allowing for detailed structural and dynamic studies of single cells, require fluorescent tags to visualize cellular architecture and may cause short- and long-term photo-damage. On the other hand, traditional light microscopy, although relatively non-invasive, does not provide detailed structural information. Optical coherence microscopy (OCM) is a promising alternative, as it does not require sample pre-processing or labelling and can provide 3D images of intracellular structures. Here we applied OCM to assess the chromatin conformation of immature mouse oocytes, a feature that corresponds with their transcriptional status and developmental competence and cannot be examined by traditional light microscopy. We showed that OCM distinguished oocytes with so-called non-surrounded nucleoli (NSN) and surrounded nucleoli (SN) chromatin conformation with very high sensitivity and specificity and that OCM scanning did not decrease the quality of oocytes. Finally, we cross-referenced OCM data with the oocyte ability to undergo normal nuclear and cytoplasmic maturation and proven that indeed oocytes scored with OCM as NSN mature less effectively than oocytes scored as SN. Our results suggest that OCM may be a valuable addition to the imaging toolkit used in assisted reproduction procedures.

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Lena Arévalo, Gina Esther Merges, Simon Schneider, and Hubert Schorle

In brief

Protamines package and shield the paternal DNA in the sperm nucleus and have been studied in many mouse models over decades. This review recapitulates and updates our knowledge about protamines and reveals a surprising complexity in protamine function and their interactions with other sperm nuclear proteins.

Abstract

The packaging and safeguarding of paternal DNA in the sperm cell nucleus is a critical feature of proper sperm function. Histones cannot mediate the necessary hypercondensation and shielding of chromatin required for motility and transit through the reproductive tracts. Paternal chromatin is therefore reorganized and ultimately packaged by protamines. In most mammalian species, one protamine is present in mature sperm (PRM1). In rodents and primates among others, however, mature sperm contain a second protamine (PRM2). Unlike PRM1, PRM2 is cleaved at its N-terminal end. Although protamines have been studied for decades due to their role in chromatin hypercondensation and involvement in male infertility, key aspects of their function are still unclear. This review updates and integrates our knowledge of protamines and their function based on lessons learned from mouse models and starts to answer open questions. The combined insights from recent work reveal that indeed both protamines are crucial for the production of functional sperm and indicate that the two protamines perform distinct functions beyond simple DNA compaction. Loss of one allele of PRM1 leads to subfertility whereas heterozygous loss of PRM2 does not. Unprocessed PRM2 seems to play a distinct role related to the eviction of intermediate DNA-bound proteins and the incorporation of both protamines into chromatin. For PRM1, on the other hand, heterozygous loss leads to strongly reduced sperm motility as the main phenotype, indicating that PRM1 might be important for processes ensuring correct motility, apart from DNA compaction.

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Huan Yin, Suye Suye, Zhixian Zhou, Haiyi Cai, and Chun Fu

In brief

Fanconi anemia results in subfertility and primary ovarian deficiency in females. This study reveals that disrupted meiosis in oocytes is one of the mechanisms involved.

Abstract

Fance is an important factor participating in the repair of DNA interstrand cross-links and its defect causes severe follicle depletion in female mice. To explore the underlying mechanisms, we investigated the effects of Fance on ovarian development in embryonic and newborn mice. We found that the number of oocytes was significantly decreased in Fance −/− mice as early as 13.5 days post coitum (dpc). The continuous decrease of oocytes in Fance −/− mice compared with the Fance +/+ mice led to the primordial follicles being almost exhausted at 2 days postpartum (dpp). The mitotic–meiotic transition occurred normally, but the meiotic progression was arrested in pachytene in Fance −/− oocytes. We detected the expressions of RAD51 (homologous recombination repair factor), 53BP1 (non-homologous end-joining repair factor), and γH2AX by immunostaining analysis and chromosome spreads. The expressions of 53BP1 were increased and RAD51 decreased significantly in Fance −/− oocytes compared with Fance +/+ oocytes. Also, the meiotic crossover indicated by MLH1 foci was significantly increased in Fance −/− oocytes. Oocyte proliferation and apoptosis were comparable between Fance −/− and Fance +/+ mice (P > 0.05). The aberrant high expression at 17.5 dpc and low expressions at 1 and 2 dpp indicated that the expression pattern of pluripotent marker OCT4 (POU5F1) was disordered in Fance −/− oocytes. These findings elucidate that Fance mutation leads to a progressive reduction of oocytes and disrupts the progression of meiotic prophase I but not the initiation. And, our study reveals that the potential mechanisms involve DNA damage repair, meiotic crossover, and pluripotency of oocytes.

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Vineet K Maurya, Maria M Szwarc, Rodrigo Fernandez-Valdivia, David M Lonard, Song Yong, Niraj Joshi, Asgerally T Fazleabas, and John P Lydon

Although a non-malignant gynecological disorder, endometriosis displays some pathogenic features of malignancy, such as cell proliferation, migration, invasion and adaptation to hypoxia. Current treatments of endometriosis include pharmacotherapy and/or surgery, which are of limited efficacy and often associated with adverse side effects. Therefore, to develop more effective therapies to treat this disease, a broader understanding of the underlying molecular mechanisms that underpin endometriosis needs to be attained. Using immortalized human endometriotic epithelial and stromal cell lines, we demonstrate that the early growth response 1 (EGR1) transcription factor is essential for cell proliferation, migration and invasion, which represent some of the pathogenic properties of endometriotic cells. Genome-wide transcriptomics identified an EGR1-dependent transcriptome in human endometriotic epithelial cells that potentially encodes a diverse spectrum of proteins that are known to be involved in tissue pathologies. To underscore the utility of this transcriptomic data set, we demonstrate that carbonic anhydrase 9 (CA9), a homeostatic regulator of intracellular pH, is not only a molecular target of EGR1 but is also important for maintaining many of the cellular properties of human endometriotic epithelial cells that are also ascribed to EGR1. Considering therapeutic intervention strategies are actively being developed for EGR1 and CAIX in the treatment of other pathologies, we believe EGR1 and its transcriptome (which includes CA9) will offer not only a new conceptual framework to advance our understanding of endometriosis but will also furnish new molecular vulnerabilities to be leveraged as potential therapeutic options in the future treatment of endometriosis.

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Nardhy Gomez-Lopez, Jose Galaz, Derek Miller, Marcelo Farias-Jofre, Zhenjie Liu, Marcia Arenas-Hernandez, Valeria Garcia-Flores, Zachary Shaffer, Jonathan M Greenberg, Kevin R Theis, and Roberto Romero

In brief

The syndrome of preterm labor comprises multiple established and novel etiologies. This review summarizes the distinct immune mechanisms implicated in preterm labor and birth and highlights potential strategies for its prevention.

Abstract

Preterm birth, the leading cause of neonatal morbidity and mortality worldwide, results from preterm labor, a syndrome that includes multiple etiologies. In this review, we have summarized the immune mechanisms implicated in intra-amniotic inflammation, the best-characterized cause of preterm labor and birth, as well as novel etiologies non-associated with intra-amniotic inflammation (i.e. formally known as idiopathic). While the intra-amniotic inflammatory responses driven by microbes (infection) or alarmins (sterile) have some overlap in the participating cellular and molecular processes, the distinct natures of these two conditions necessitate the implementation of specific approaches to prevent adverse pregnancy and neonatal outcomes. Intra-amniotic infection can be treated with the correct antibiotics, whereas sterile intra-amniotic inflammation could potentially be treated by administering a combination of anti-inflammatory drugs (e.g. betamethasone, inflammasome inhibitors, etc.). Recent evidence also supports the role of fetal T-cell activation as a newly described trigger for preterm labor and birth in a subset of cases diagnosed as idiopathic. Moreover, herein we also provide evidence of two maternally-driven immune mechanisms responsible for preterm births formerly considered to be idiopathic. First, the impairment of maternal Tregs can lead to preterm birth, likely due to the loss of immunosuppressive activity resulting in unleashed effector T-cell responses. Secondly, homeostatic macrophages were shown to be essential for maintaining pregnancy and promoting fetal development, and the adoptive transfer of homeostatic M2-polarized macrophages shows great promise for preventing inflammation-induced preterm birth. Collectively, in this review, we discuss the established and novel immune mechanisms responsible for preterm birth and highlight the potential targets for novel strategies aimed at preventing the multi-etiological syndrome of preterm labor leading to preterm birth.

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Robert John Aitken, Elizabeth Bromfield, and Zamira Gibb

This article lays out the fundamental principles of oxidative stress. It describes the nature of reactive oxygen species (ROS), the way in which these potentially toxic metabolites interact with cells and how they impact both cellular function and genetic integrity. The mechanisms by which ROS generation is enhanced to the point that the cells’ antioxidant defence mechanisms are overwhelmed are also reviewed taking examples from both the male and female reproductive system, with a focus on gametogenesis and fertilization. The important role of external factors in exacerbating oxidative stress and impairing reproductive competence is also examined in terms of their ability to disrupt the physiological redox regulation of reproductive processes. Developing diagnostic and therapeutic strategies to cope with oxidative stress within the reproductive system will depend on the development of a deeper understanding of the nature, source, magnitude, and location of such stress in order to fashion personalized treatments that meet a given patient’s clinical needs.

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Satoshi Funaya, Yuria Kawabata, Kenta Sugie, Ken-ichiro Abe, Yutaka Suzuki, Masataka G Suzuki, and Fugaku Aoki

In brief

In oocytes, chromatin structure is loosened during their growth, which seems to be essential for the establishment of competence to accomplish the maturation and further development after fertilization. This paper shows that a linker histone variant, H1foo, is involved in the formation of loosened chromatin structure in growing oocytes.

Abstract

During oogenesis, oocytes show a unique mode of division and gene expression patterns. Chromatin structure is thought to be involved in the regulation of these processes. In this study, we investigated the functions of linker histones, which modulate higher-order chromatin structure during oogenesis. Because H1foo is highly expressed in oocytes, we knocked down H1foo using siRNA and observed oocyte growth, maturation, and fertilization. However, H1foo knockdown had no effect on any of these processes. Overexpression of H1b or H1d, which has a high ability to condense chromatin and is expressed at a low level in oocytes, resulting in tightened chromatin and a decreased success rate of oocyte maturation. By contrast, overexpression of H1a, which is expressed at a high level in oocytes and has a low ability to compact chromatin, did not affect growth or maturation. Therefore, H1a, but not other variants, might compensate for the function of H1foo in H1foo-knockdown oocytes. These results implicate H1foo in the formation of loose chromatin structure, which is necessary for oocyte maturation. In addition, the low expression of somatic linker histone variants, for example, H1b and H1d, is important for loosened chromatin and meiotic progression.