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Pascal Adam, Laurence Tardif, François Fabi, Dadou Likonza Lokengo, Léa-Isabelle Renaud, Sophie Parent, Monique Cadrin, Derek Boerboom, and Eric Asselin

In brief

The regulation of AKT in the endometrium during many cellular processes such as apoptosis and cell survival is crucial during the estrous cycle to ensure fertility. This research shows the specific function of AKT isoforms in the mouse endometrium for litter size, estrous cyclicity and endometrial gland development.

Abstract

Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation, and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation is differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in progesterone receptor-expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest not only specific roles between each isoform but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.

Open access

I T Harris, C Maddock, M Farnworth, K Nankervis, J Perrett, A Z Pyatt, and R N Blanchard

In brief

Adverse trends in reproductive function are a concern in humans, companion, livestock, and wildlife species. This study indicates that equine populations are at risk of a comparable decline in sperm progressive motility.

Abstract

There is increasing evidence reporting geographically sensitive adverse trends in human semen quality, with parallel trends observed in the dog sentinel. Despite significant economic and welfare complications associated with poor testicular function, trends in current equine populations are undetermined. Given the predictive value of sperm progressive motility (PMOT) in male factor infertility and fertilisation potential, research determining trends in this parameter is warranted. This research analysed trends in stallion sperm PMOT through systematic review and meta-regression. Using a comprehensive search strategy, Scopus, Embase (Ovid), Medline (Ovid), and VetMed (CAB direct) were scoped for eligible data. Using best practices, 230 meta-data points from 229 articles published from 1991 to 2021 were collated for meta-regression analysis. Sperm PMOT declined significantly between 1984 and 2019 (simple linear regression: b −0.340, P = 0.017; meta-regression: b −0.610, P ≤ 0.001). Overall and yearly PMOT declines were predicted at 33.51 and 0.96%, respectively (1984: 63.69 ± 5.07%; 2019: 42.35 ± 3.69%). Trends remained consistent irrespective of sensitivity analyses. Yearly and overall declines were stronger in western (yearly: 0.75%, overall: 26.29%) compared to non-western (yearly: 0.46%, overall: 10.65%) populations. Adverse trends contribute vital data to the debate surrounding declining semen quality, supporting the use of equines as novel comparative models for human reproduction. Results could have significant economic, health, and welfare consequences for equine breeding sectors. A comparable decline in human, dog, and horse sperm quality is indicative of a common environmental aetiology, indicating the need for a holistic One Health approach in determining causes and developing preventative strategies.

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Zachary K Seekford, Dylan B Davis, Mackenzie J. Dickson, Lucas Melo Goncalves, Samir Burato, Matthew P. Holton, Julie Gordon, Ky G. Pohler, G. Cliff Lamb, Timothy D. Pringle, Robert L. Stewart, Maria S. Ferrer, Pedro Fontes, and John J Bromfield

Bulls used in cattle production are often overfed to induce rapid growth, early puberty and increase sale price. While the negative consequences of undernutrition on bull sperm quality are known, it is unclear how a high gain diet influences embryo development. We hypothesized that semen collected from bulls fed a high gain diet would have a reduced capacity to produce blastocysts following in vitro fertilization. Eight mature bulls were stratified by body weight and fed the same diet for 67 d at either a maintenance level (0.5% body weight per day; n = 4) or a high gain rate (1.25% body weight per day; n = 4). Semen was collected by electroejaculation at the end of the feeding regimen and subjected to sperm analysis, frozen, and used for in vitro fertilization. The high gain diet increased body weight, average daily gain, and subcutaneous fat thickness compared to the maintenance diet. Sperm of high gain bulls tended to have increased early necrosis and had increased post-thaw acrosome damage compared with maintenance bulls, but diet did not affect sperm motility or morphology. Semen of high gain bulls reduced the percentage of cleaved oocytes that developed to blastocyst stage embryos. Paternal diet had no effect on the number of total or CDX2 positive cells of blastocysts, or blastocysts gene expression for markers associated with developmental capacity. Feeding bulls a high gain diet did not affect sperm morphology or motility, but increased adiposity and reduced the ability of sperm to generate blastocyst stage embryos.

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Beatriz Macías-García and Lauro González-Fernández

In brief

The mechanism by which p32 protein increases during capacitation in boar spermatozoa is unknown. This manuscript shows a new mechanism of induction of p32 in boar spermatozoa: the proteolysis of the phosphorylated and glycosylated form of SPACA1.

Abstract

Protein tyrosine phosphorylation (PY) induction is associated with sperm capacitation. We previously showed that calcium-sensing receptor (CASR) inhibition by NPS2143 induces the 32 kDa tyrosine-phosphorylated protein (p32) in boar spermatozoa. We showed that NPS2143 induced an increase in p32 and loss of acrosomal integrity in live and dead spermatozoa in capacitating conditions (Tyrode's complete medium); the p32 rise occurred in dead spermatozoa, as shown by flow cytometry sorting. EGTA addition blunted the increase in p32, the loss of acrosomal integrity, and the increase in dead spermatozoa induced by NPS2143, indicating that the effects of NPS2143 are calcium-dependent. Mass spectrometry was used to identify which tyrosine-phosphorylated proteins were induced by NPS2143, but only serine/threonine-phosphorylated proteins were found; among these, SPACA1 was identified with different molecular weights (18, 32, and 35–45 kDa). We confirmed tyrosine phosphorylation of SPACA1 at 32 and 35–45 kDa by immunoprecipitation and co-localization of PY and SPACA1 in the presence of NPS2143 by immunofluorescence. The molecular weight of SPACA1 (35–45 kDa) decreased after treatment with peptide-N-glycosidase F, indicating that this protein is N-glycosylated. The soybean trypsin inhibitor (STI), a serine protease inhibitor, suppressed the appearance of p32 and SPACA1 (30 and 32 kDa) induced by NPS2143. Also, 8-Br-cAMP and A23187 treatments induced an increase in p32 and SPACA1 (30–32 kDa) and a parallel induction of the acrosome reaction. These findings suggest that CASR inhibition induces loss of acrosomal integrity and proteolysis of the glycosylated and phosphorylated SPACA1 (35–45 kDa) resulting in a SPACA1 rise at 32 kDa (p32).

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Joao de Agostini Losano, Christopher Souders, Christopher Martyniuk, and Bradford William Daigneault

Sperm transport through the female reproductive tract requires energy for motility and fertilization. Sperm kinematic assessment is conducted as an industry standard to estimate semen quality prior to bovine insemination. However, individual samples with similar post-thaw motility result in different pregnancy outcomes, suggesting that differences in bioenergetics may be important for sperm function. Thus, characterization of bioenergetic and kinematic parameters of sperm over time may reveal novel metabolic requirements for sperm function. Post-thawed sperm from five samples of individual (A, B, C) and pooled bulls (AB, AC) were assessed at 0 and 24 hr after thawing. Sperm were evaluated for kinematics via Computer-Assisted Sperm Analyses and bioenergetic profiles using a Seahorse Analyzer for basal respiration [BR], mitochondrial stress test [MST] and energy map [EM]. Motility was nearly identical among samples after thawing and no differences in bioenergetics were detected. However, after 24 hr of sperm storage, pooled sperm samples (AC) presented with higher BR and proton leakage compared to other samples. Sperm kinematic variability among samples was higher after 24 hr, suggesting difference in sperm quality may manifest over time. Despite a reduction in motility and mitochondrial membrane potential, BR was higher at 24 hr compared to 0 hr for nearly all samples. A metabolic divergence between samples was detected by EM, indicating a shift in bioenergetic profiles over time that was undetected after thawing. These new bioenergetic profiles elucidate a novel dynamic plasticity of sperm metabolism over time while suggesting an influence of heterospermic interactions for further investigation.

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Teruhito Ishihara, Jane C Fenelon, Oliver W Griffith, Kei-ichiro Ishiguro, and Marilyn B Renfree

In brief

Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other.

Abstract

In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

Open access

Jiangman Gao, Zhiqiang Yan, Liying Yan, Xiaohui Zhu, Hui Jiang, and Jie Qiao

We aimed to investigate the correlation of sperm DNA fragmentation (SDF) with the incidence and paternal origin of whole and segmental chromosomal aneuploidies of embryos at the blastocyst stage. A retrospective cohort study was conducted with a total of 174 couples (women aged 35 years or younger) who underwent 238 cycles (including 748 blastocysts) of preimplantation genetic testing for monogenic diseases (PGT-M). All subjects were divided into two groups based on the sperm DNA fragmentation index (DFI) level: low DFI (<27%) and high DFI (≥27%). The rates of euploidy, whole chromosomal aneuploidy, segmental chromosomal aneuploidy, mosaicism, parental origin of aneuploidy, fertilization, cleavage, and blastocyst formation were compared between low and high DFI groups. We found no significant differences in fertilization, cleavage, or blastocyst formation between the two groups. Compared to that in the low DFI group, segmental chromosomal aneuploidy rate was significantly higher in the high DFI group (11.57 vs. 5.83%, P=0.021; OR 2.32, 95% CI 1.10-4.89, P=0.028). The whole chromosomal embryonic aneuploidy of paternal origin was significantly higher in cycles with high DFI than in cycles with low DFI (46.43% vs. 23.33%, P=0.018; OR 4.32, 95% CI 1.06-17.66, P=0.041). However, the segmental chromosomal aneuploidy of paternal origin was not significantly different between the two groups (71.43% vs. 78.05%, P=0.615; OR 1.01, 95% CI 0.16-6.40, P=0.995). In conclusion, our results suggested that high sperm DNA fragmentation was associated with the incidence of segmental chromosomal aneuploidy and increased paternal whole chromosomal aneuploidies in embryos.

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Thales de Souza França, Itamar Cossina Gomes, Eduardo Antônio Sanches, Maritza Pérez Atehortúa, Nathalia Dos Santos Teixeira, Rômulo Batista Rodrigues, Thaiza Rodrigues De Freitas, Jhony Lisboa Benato, Lis Santos Marques, Ana Regina Seabra de Souza, and Danilo Streit Jr

Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from twelve South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

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Suye Suye, Huan Yin, Zhixian Zhou, Chunying Zheng, Zhen Ren, Liye Shi, and Chun Fu

Primordial germ cells (PGCs) development is a subtle and complex regulatory process. Fance is an important substrate molecule necessary for the activation of the Fanconi anemia (FA) pathway, and its homozygous mutant causes massive oocyte loss as early as embryonic day 13.5 (E13.5). Here, we present histological and RNA-seq analysis of Fance deficient PGCs to explore the possible mechanisms responsible for its progressive depletion of germ cells. In Fance-/- embryos, the reduction of PGCs was already evident at E9.5 and the progressive loss of PGCs led to the PGCs being almost exhausted at E12.5. An increase of apoptotic cells was detected among Fance-/- PGCs, which may intuitively explain their reduced number in embryos. Moreover, abnormal cell proliferation and accumulating DNA damage were detected in E12.5 Fance-/- PGCs. We identified 3026 differentially expressed genes in E12.5 Fance-/- PGCs compared to Fance+/+ . KEGG pathway analysis revealed that the up-regulated genes were highly associated with ‘lysosome’, various metabolism pathways, whereas the down-regulated genes were mainly enriched in ‘cell cycle’, ‘oocyte meiosis’, ‘ribosome’, and various DNA repair pathways. In addition, multiple genes of various cell death pathways were found to be differentially expressed in E12.5 Fance-/- PGCs, indicating that PGCs death in Fance-/- embryos might diverge from canonical apoptosis. These findings indicate that Fance is essential for PGCs survival and the potential mechanisms involve cell cycle regulation, DNA damage repair, cell death prevention, and by regulating lysosome and ribosome function. Our results provide an important reference for further studies.

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Xue-Yun Qin, Hui-Hui Shen, Xin-Yan Zhang, Xing Zhang, Feng Xie, Wen-Jun Wang, Yu Xiong, Jie Mei, and Ming-Qing Li

In brief

Hypoxia is vital for the establishment of the maternal–fetal interface during early pregnancy. This study shows that decidual macrophages (dMφ) could be recruited and reside in decidua under the regulation of hypoxia/VEGFA-CCL2 axis.

Abstract

Infiltration and residence of decidual macrophages (dMφ) are of great significance to pregnancy maintenance for their role in angiogenesis, placental development, and inducing immune tolerance. Besides, hypoxia has now been acknowledged as an important biological event at maternal–fetal interface in the first trimester. However, whether and how hypoxia regulates biofunctions of dMφ remain elusive. Herein, we observed increased expression of C–C motif chemokine ligand 2 (CCL2) and residence of macrophages in decidua compared to secretory-phase endometrium. Moreover, hypoxia treatment on stromal cells improved the migration and adhesion of dMφ. Mechanistically, these effects might be mediated by upregulated CCL2 and adhesion molecules (especially ICAM2 and ICAM5) on stromal cells in the presence of endogenous vascular endothelial growth factor-A (VEGFA) in hypoxia. These findings were also verified by recombinant VEGFA and indirect coculture, indicating that the interaction between stromal cells and dMφ in hypoxia condition may facilitate dMφ recruitment and residence. In conclusion, VEGFA derived from a hypoxic environment may manipulate CCL2/CCR2 and adhesion molecules to enhance the interactions between dMφ and stromal cells and thus contribute to the enrichment of macrophages in decidua early during normal pregnancy.