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Mitochondria are important organelles in eukaryotic cells and play an essential role in energy production and cell signaling. However, the importance of mammalian sperm mitochondria as an energy source remains to be elucidated because glycolysis is known to be dominant. In this context, one of the functions of mammalian sperm mitochondria is considered as a calcium ion (Ca2+) homeostasis. Previously, the Ca2+ level within the mitochondria of mouse sperm under resting conditions was reported to be high (in the micromolar range) using the fluorescent Ca2+ indicator Calcium Green-5N (CG-5N). To confirm this fact, we performed the semi-quantitative determination of Ca2+ concentration with several Ca2+ indicators. Although we reproduced the previous report of CG-5N, other Ca2+ indicators do not support the result obtained with CG-5N. The results obtained with Rhod-2, Fluo-3, and Fluo-5N indicate that the free Ca2+ concentration in mitochondria is comparable to that of the cytosol at the resting condition and under the condition stimulated by ATP. Although we still do not understand why CG-5N exhibits a distinct result from other indicators, the regulation of Ca2+ concentration in murine sperm mitochondria is analogous to that observed in somatic cells. Namely, the Ca2+ concentrations within sperm mitochondria fluctuate in response to changes in cytosolic Ca2+ levels. Our results contribute to a revised understanding of the role of mitochondria in Ca2+ homeostasis in mammalian sperm.
Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China
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Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China
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In brief
Preeclampsia (PE) is a severe complication that leads to major maternal and fetal mortality and morbidity, and one of its causes is extravillous trophoblast (EVT) dysfunction. This study revealed the role of CD74 in the invasion and proliferation of EVTs.
Abstract
PE is a severe hypertensive disorder during pregnancy, and one of its causes is the dysfunction of EVTs. In this study, we analyzed single-cell RNA sequencing (scRNA-seq) data of placentas from PE patients and the sirtuin 1 (SIRT1) heterozygous knockout mouse model, which exhibited typical PE-like symptoms. We identified 134 differentially expressed genes (DEGs) with similar trends in EVTs of PE patients and in parietal trophoblast giant cells (P-TGCs) of Sirt1−/− (HO) placentas from Sirt1+/− (HE) pregnant mice. Interestingly, Kyoto Encyclopedia of Genes and Genomes analysis showed that 134 overlapping genes were related to the MAPK signaling pathway. We validated several DEGs using immunofluorescence at the protein level. Finally, we selected CD74 for further experiments, which showed a decrease in EVTs of PE patients and in P-TGCs of Sirt1−/− placentas from Sirt1+/− pregnant mice. Additionally, cell proliferation assays and transwell assays showed that the proliferation and invasion abilities were decreased in CD74 knockdown HTR8/SVneo cells using lentivirus transfection, which can be improved by adding the SIRT1 agonist SRT1720 or metformin, an agonist of the MAPK signaling pathway. Importantly, the expression of CD74 can be positively regulated by SIRT1. These data suggest that CD74 plays an important protective role in the pathogenesis of preeclampsia by regulating the MAPK signaling pathway, which can be regulated by SIRT1.
Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC, Canada
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In brief
Ubiquitination plays a pivotal role in a multitude of cellular functions; however, the precise contributions of various ubiquitin ligases in governing early developmental processes remain largely unexplored. This study revealed that the E3 ubiquitin ligases DCAF13 and RNF114 are both necessary for the normal regulation of early porcine embryo development.
Abstract
Ubiquitylation is required for normal regulation of many biological functions by modulating several protein facets such as structure, stability, interaction, localization, and degradation. In this study, we explored the roles of two E3 ubiquitin ligases (E3s), the DDB1- and CUL4-associated factor 13 (DCAF13) and the Ring finger protein 114 (RNF114), in the regulation of porcine embryo development. Attenuation of DCAF13 mRNA decreased embryo development at the blastocyst stage, while the development of RNF114-attenuated embryos was not significantly different than that of control embryos. The average number of cells per blastocyst was decreased in DCAF13-attenuated embryos and increased in RNF114-attenuated embryos compared to controls. The relative mRNA abundance of the histone methyltransferase SUV39H1, which regulates histone H3 lysine 9 trimethylation (H3K9me3), was increased in both DCAF13- and RNF114-attenuated embryos, but nuclear immunofluorescence signal for H3K9me3 on day 3 embryos was not significantly altered between attenuated and control embryos. Nuclear immunofluorescence signal for H3K4m3 was decreased in DCAF13-attenuated embryos, but it was increased in RNF114-attenuated embryos compared to controls. Attenuation of DCAF13 and RNF114 mRNAs increased transcript levels for the DNA recombinase RAD51 and decreased expression of phosphorylated histone H2A.X (γH2AX), which suggests an impact on DNA damage repair. In addition, lower mRNA expression of the lysine demethylases 5B (KDM5B) and 5C (KDM5C), both involved in embryo genome activation and DNA repair, was detected in DCAF13-attenuated embryos. These findings indicated that both DCAF13 and RNF114 have important roles in the regulation of the early development of porcine embryos.
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In brief
Genistein contributes to granulosa cell (GC) survival by two routes: one is that genistein induced p-AMPK and inhibited p-mTOR, which induces LC3 activation and autophagy; the other is that genistein inhibited caspase-3 and its cleavage, which induces PARP1 activation and PARylation.
Abstract
Genistein is an isoflavone which is beneficial for health, but little is known regarding its function on granulosa cell fate during follicular atresia. In the present study, we established an in vitro model of porcine follicular granulosa cell apoptosis by serum deprivation and showed that treatments with 1 μM and 10 μM genistein significantly reduced the apoptotic rate of granulosa cells compared to the blank control (P < 0.05). These results suggest that genistein at micromolar levels alleviates serum deprivation-induced granulosa cell apoptosis, and the ameliorative effect of genistein on granulosa cell apoptosis is likely to be able to inhibit nutrient depletion-induced follicular atresia. Further experimental results revealed that the expression of the autophagic marker protein LC3II in 100 nM–10 μM genistein treatment increased in a dose-dependent manner and was higher than the control (P < 0.05). Genistein also dose dependently promoted the phosphorylation of AMPK (adenosine 5’-monophosphate-activated protein kinase) in granulosa cells. Poly(ADP-ribose) (pADPr) formation in genistein-treated groups was also notably higher than in the controls (P < 0.05). Collectively, genistein alleviates serum deprivation-induced granulosa cells in vitro through enhancing autophagy, which involving AMPK activation and PARylation signaling. However, further study should be carried out to investigate the role of the aforementioned signaling on this process.
Graduate School of Agriculture, Hokkaido University, Sapporo, Japan
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In brief
Interferon tau (IFNT) stimulates lysosomal activation via the Janus-activated kinase in peripheral blood leukocytes during pregnancy recognition. IFNT-mediated lysosomal activation could serve as a novel marker for early pregnancy in cattle.
Abstract
IFNT is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post artificial insemination, after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein (LAMP) 1, 2, CTSB and CTSK. Expression of all genes was significantly higher in PBLs of pregnant cows than in nonpregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.
Freie Universitat Berlin, Veterinary Medicine, Berlin, Germany
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Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
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Universita degli Studi di Padova, Ethics Laboratory for Veterinary Medicine, Conservation, and Animal Welfare, Veneto, Padova, Italy
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Universita degli Studi di Padova, Ethics Laboratory for Veterinary Medicine, Conservation, and Animal Welfare, Veneto, Padova, Italy
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Fondazione Avantea, Riproduzione Cremona, Lombardy, Cremona, Italy
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In brief
To save endangered rhinoceros species, assisted reproductive technologies are warranted. We here report in vitro blastocyst generation of the Near-Threatened Southern white rhinoceros and, for the first time, also of the technically Extinct Northern white rhinoceros.
Abstract
The Anthropocene is marked by a dramatic biodiversity decline, particularly affecting the family Rhinocerotidae. Three of five extant species are listed as Critically Endangered (Sumatran, Javan, black rhinoceros), one as Vulnerable (Indian rhinoceros), and only one white rhino (WR) subspecies, the Southern white rhinoceros (SWR), after more than a century of successful protection is currently classified as Near Threatened by the IUCN, while numbers again are declining. Conversely, in 2008, the SWR’s northern counterpart and second WR subspecies, the Northern white rhinoceros (NWR), was declared extinct in the wild. Safeguarding these vanishing keystone species urgently requires new reproductive strategies. We here assess one such strategy, the novel in vitro fertilization program in SWR and – for the first-time NWR – regarding health effects, donor-related, and procedural factors. Over the past 8 years, we performed 65 procedures in 22 white rhinoceros females (20 SWR and 2 NWR) comprising hormonal ovarian stimulation, ovum pick-up (OPU), in vitro oocyte maturation, fertilization, embryo culture, and blastocyst cryopreservation, at an efficiency of 1.0 ± 1.3 blastocysts per OPU, generating 22 NWR, 19 SWR and 10 SWR/NWR hybrid blastocysts for the future generation of live offspring.
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This study investigated if oocyte and early embryo epigenetic programming are dependent on oocyte mitochondrial ATP production. A bovine in vitro experiment was performed in which oocyte mitochondrial ATP production was reduced using 5nmol/l oligomycin A (OM; ATP synthase inhibitor) during in vitro maturation (IVM) compared to control (CONT). OM-exposure significantly reduced mitochondrial ATP production rate in MII oocytes (34.6% reduction, P=0.018) and significantly decreased embryo cleavage rate at 48h p.i. (7.6% reduction, P=0.031). Compared to CONT, global DNA methylation (5mC) levels were decreased in OM-exposed MII oocytes (9.8% reduction, P=0.019) while global histone methylation (H3K9me2) was increased (9.4% increase, P=0.024). In zygotes, OM-exposure during IVM increased 5mC (22.3% increase, P<0.001) and histone acetylation (H3K9ac, 17.3% increase, P=0.023) levels, while H3K9me2 levels were not affected. In morulae, 5mC levels were increased (10.3% increase, P=0.041) after OM-exposure compared to CONT, while there was no significant difference in H3K9ac and H3K9me2 levels. These epigenetic alterations were not associated with any persistent effects on embryo mitochondrial ATP production rate or mitochondrial membrane potential (assessed at the 4-cell stage). Also, epigenetic regulatory genes were not differentially expressed in OM-exposed zygotes or morulae. Finally, apoptotic cell index in blastocysts was increased after OM-exposure during oocyte maturation (41.1% increase, P<0.001). We conclude that oocyte and early embryo epigenetic programming are dependent on mitochondrial ATP production during IVM.
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In brief
Partially denuded mouse cumulus–oocyte complexes restore likely functional transzonal projections in culture, under meiotic inhibition, with no detectable impact on oocyte competence. This proof-of-concept study constitutes positive premises for improving the developmental competence of human capacitation (CAPA)–in vitro maturation (IVM) oocytes with inadequate somatic cell connections.
Abstract
In vitro oocyte culture might be the sole option for fertility preservation in some patients. This relies on constant oocyte–somatic bidirectional communication, and its precocious disruption alters oocyte competence. In non-human chorionic gonadotropin-triggered human in vitro maturation (IVM), retrieval of cumulus–oocyte complexes (COCs) by needle aspiration from the targeted small follicles (2–8 mm) leads to the collection of some partially denuded (PD) COCs with poor developmental competence. Hypothetically, re-establishing connectivity in these COCs could rescue oocyte quality. To test this, we used a well-characterized mouse preantral follicle culture system. On day 8, at antral stage, in part of the follicles, the oocytes were mechanically denuded while in other follicles in vitro grown oocytes were replaced with age matched fully stripped in vivo grown ones. The denuded oocytes were cultured on top of the somatic compartment until day 12, when oocyte–somatic reconnection was assessed. Furthermore, to better mimic the current biphasic IVM setup, fully surrounded (FS) COCs were collected from 19- to 21- day-old unprimed mice. Following partial mechanical denudation, COCs were cultured under meiotic inhibition for 2–4 days, to test oocyte–cumulus cell (CC) reconnection. Meiotic and developmental competence endpoints were compared between reconnected and FS-cultured COCs. We concluded that (i) in vivo- and in vitro- grown antral oocytes reconnect with in vitro-grown somatic companions; (ii) PD–COCs restore the FS morphology in culture, under meiotic inhibition; and (iii) oocyte quality from reconnected and intact cultured COCs is comparable. These observations encourage translational work to rescue partially denuded oocytes in human IVM.
The Jackson Laboratory, Bar Harbor, Maine, USA
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Biochemistry and Molecular Pharmacology Department, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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In brief
A new allele of the senataxin gene Setx spcar3 causes meiotic arrest of spermatocytes with aberrant DNA damage and accumulation of R-loops.
Abstract
An unbiased screen for discovering novel mouse genes for fertility identified the spcar3, spermatocyte arrest 3, mutant phenotype. The spcar3 mutation identified a new allele of the Setx gene, encoding senataxin, a DNA/RNA helicase that regulates transcription termination by resolving DNA/RNA hybrid R-loop structures. The Setxspcar3 mutant mice exhibit male infertility and female subfertility. Histology of the Setxspcar3 mutant testes revealed the absence of spermatids and mature spermatozoa in the seminiferous tubules. Cytological analysis of chromosome preparations of the Setxspcar3 mutant spermatocytes revealed normal synapsis, but aberrant DNA damage in the autosomes, defective formation of the sex body, and arrest of meiosis in mid-prophase. Additionally, Setxspcar3 testicular cells exhibit abnormal accumulation of R-loops. Transient expression assays identified regions of the senataxin protein required for sub-nuclear localization. Together, these results not only confirm that senataxin is required for normal meiosis and spermatogenesis but also provide a new resource for the determination of its role in maintaining R-loop formation and genome integrity.
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In brief
Capacitation is regulated by decapacitation factors secreted by male ducts and accessory sex glands. This revision is focused on targets and events regulated by decapacitation factors in Mus musculus and their potential use for fertility control.
Abstract
Sperm capacitation is a necessary process for mammalian spermatozoa to acquire fertilization capability. This process occurs when the sperm enters the female’s reproductive duct, involving a vital interplay with the uterine and oviductal environment, leading to morphological, physiological, and biochemical modifications in the male gamete. Besides, for a successful sperm capacitation, molecules are incorporated onto the sperm’s surface during its passage through the male reproductive tract followed by their subsequent removal. These molecules, referred to as decapacitation factors (DFs), also regulate capacitation, preventing this process from occurring in the wrong site or at the wrong time. While decapacitation factors have been extensively studied in recent decades in species such as Mus musculus, there is no comprehensive report consolidating information on all the identified decapacitation factors and the molecular basis of their function. The aim of this review is to summarize the data related to decapacitation factors discovered and characterized in Mus musculus. Concurrently, this review aims to elucidate the implications of different decapacitation factors throughout the fertilization process (i.e. capacitation, acrosomal reaction, and fertilization), as well as the methodologies employed for their investigation. Given that mice (Mus musculus) have served as a valuable model in reproductive research due to their genetic similarity to humans, this review contributes to our understanding of the role of decapacitation factors in male fertility.
