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Modulatory role of mTOR in trophoblast adaptive response in the early stage of placentation in sheep

I Viola, P Toschi, I Manenti, P Accornero, and M Baratta

In brief

Fibroblast growth factor-2 (FGF2) is essential for early placenta development in sheep. This study shows that the mechanistic target of rapamycin is the key modulator of trophoblast adaptive response under FGF2 modulation.

Abstract

During the early stage of placentation in sheep, normal conceptus development is affected by trophoblast cell functionality, whose dysregulation results in early pregnancy loss. Trophoblast metabolism is supported mainly by histotrophic factors, including fibroblast growth factor-2 (FGF2), which are involved in cell differentiation and function through the modulation of specific cellular mechanisms. The mechanistic target of rapamycin (mTOR) is known as a cellular ‘nutrient sensor’, but its downstream regulation remains poorly understood. The hypothesis was that during trophoblast development, the FGF2 effect is mediated by mTOR signalling pathway modulation. Primary trophoblast cells from 21-day-old sheep placenta were characterised and subjected to FGF2 and rapamycin treatment to study the effects on cell functionality and gene and protein expression profiles. The model showed mainly mononuclear cells with epithelial cell-like growth and placental morphological properties, expressing typical trophoblast markers. FGF2 promoted cell proliferation and migration under normal culture conditions, whereas mTOR inhibition reversed this effect. When the mTOR signalling pathway was activated, FGF2 failed to influence invasion activity. mTOR inhibition significantly reduced cell motility, but FGF2 supplementation restored motility even when mTOR was inhibited. Interestingly, mTOR inhibition influenced endocrine trophoblast marker regulation. Although FGF2 supplementation did not affect ovine placenta lactogen expression, as observed in the control, interferon-tau was drastically reduced. This study provides new insights into the mechanism underlying mTOR inhibitory effects on trophoblast cell functionality. In addition, as mTOR is involved in the expression of hormonal trophoblast markers, it may play a crucial role in early placenta growth and fetal–maternal crosstalk.

DOI:
https://doi.org/10.1530/REP-22-0356
Volume 165: Issue 3,
Pages:
313–324 (12 total)
Restricted access

Mouse uterine stem cells are affected by endocrine disruption and initiate uteropathies

Pushpa Singh and Deepa Bhartiya

In brief

Incidence of uteropathies has increased in recent times, possibly due to exposure to endocrine-disrupting chemicals during early development. The present study shows that various uteropathies like endometrial cancer, adenomyosis, and endometriosis are interlinked and occur due to the dysfunction of tissue-resident, very small embryonic-like stem cells (VSELs).

Abstract

Underlying pathomechanisms leading to the initiation of uteropathies including non-receptive endometrium, hyperplasia, adenomyosis, endometriosis, fibroids, and cancer remain elusive. Two populations of stem cells exist in mouse uterus including pluripotent VSELs and ‘progenitors’ termed endometrial stem cells (EnSCs) which express ERα, ERβ, PR, and FSHR, participate in the regular remodelling, and maintain life-long homeostasis. The present study aimed to delineate possible stem cell origins for various uteropathies. For this, mouse pups were treated with oestradiol or diethylstilbestrol and were studied for adult onset of various uteropathies. Treatment resulted in disrupted oestrous cycles, reduced uterine weights, and marked hyperplasia in both epithelial and myometrial compartments, and the stromal compartment was also affected. VSELs were increased in numbers as judged by flow cytometry and increased expression of transcripts specific for Oct-4A, Sox-2, and Nanog, but their further differentiation into a receptive endometrium was affected. Reduced 5-methyl cytosine expression suggested global hypomethylation and was associated with several oncogenic events including loss of tumour-suppressor genes (Pten, p53), dysregulated DNA mismatch repair axis, and repair enzymes. Stem cells were epigenetically altered and showed increased expression of DNMTs, loss of imprinting loci (Igf2-H19, Dlk1-Meg3), and Ezh2. Increased co-expression of CD166 and ALDHA1 with OCT-4 in stem cells was associated with increased Esr-2 and reduced Pr in the endometrium, while both were several folds upregulated in the myometrium. Study results suggest that various uteropathies ensue due to the dysfunction of tissue-resident stem cells and provide huge scope for further research.

DOI:
https://doi.org/10.1530/REP-22-0337
Volume 165: Issue 3,
Pages:
249–268 (20 total)
Free access

Omne vivum ex ovo: the oocyte reprogramming and remodeling activities

Helena Fulka, Pasqualino Loi, Marta Czernik, Azim Surani, and Josef Fulka

In brief

Understanding the establishment of post-fertilization totipotency has broad implications for modern biotechnologies. This review summarizes the current knowledge of putative egg components governing this process following natural fertilization and after somatic cell nuclear transfer.

Abstract

The mammalian oocyte is a unique cell, and comprehending its physiology and biology is essential for understanding fertilization, totipotency and early events of embryogenesis. Consequently, research in these areas influences the outcomes of various technologies, for example, the production and conservation of laboratory and large animals with rare and valuable genotypes, the rescue of the species near extinction, as well as success in human assisted reproduction. Nevertheless, even the most advanced and sophisticated reproductive technologies of today do not always guarantee a favorable outcome. Elucidating the interactions of oocyte components with its natural partner cell – the sperm or an ‘unnatural’ somatic nucleus, when the somatic cell nucleus transfer is used is essential for understanding how totipotency is established and thus defining the requirements for normal development. One of the crucial aspects is the stoichiometry of different reprogramming and remodeling factors present in the oocyte and their balance. Here, we discuss how these factors, in combination, may lead to the formation of a new organism. We focus on the laboratory mouse and its genetic models, as this species has been instrumental in shaping our understanding of early post-fertilization events.

DOI:
https://doi.org/10.1530/REP-22-0124
Volume 165: Issue 3,
Pages:
R75–R89
Restricted access

Paradigm shift in frog sperm cryopreservation - reduced role for non-penetrating cryoprotectants

Rose Upton, Simon Clulow, Kim Colyvas, Michael Mahony, and John Clulow

Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of penetrative (PCP) and non-penetrative cryoprotectants (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide [Me2SO]) and two concentrations of a NPCP (1 and 10% w/v sucrose) in various combinations on sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.

DOI:
https://doi.org/10.1530/REP-22-0486
Accepted Manuscripts
Free access

piRNAs in sperm function and embryo viability

Giulia Perillo, Keigo Shibata, and Pei-Hsuan Wu

In brief

Mouse PIWI-interacting RNAs (piRNAs) are indispensable for spermatogenesis, but whether these small RNAs serve any function beyond gametogenesis is rarely explored. This review summarizes recent findings that demonstrated a requirement for piRNAs in sperm maturation and discusses a potential intergenerational role for paternal piRNAs.

Abstract

Unique to animals, PIWI-interacting RNAs (piRNAs) defend organisms against threats to germline integrity evoked by transposons, retroviruses, and inappropriate expression of protein-coding genes. Characterization of mouse piRNAs and studies of more than a dozen piRNA pathway protein mutants detailed in the past 15 years have firmly established an essential role for piRNAs in male fertility. Despite their vital function in spermatogenesis, mammalian piRNAs were thought to be dispensable beyond gamete formation because all piRNA pathway protein mouse mutants are invariably sterile and do not produce sperm. In contrast to the specialized purpose of piRNAs in gamete formation, tRNA-derived fragments and microRNAs have been the focus of research in RNA-mediated paternal contribution, providing additional examples of the versatility of non-coding RNAs. In recent years, the direct elimination of mouse piRNAs using CRISPR/Cas revealed their extended function in post-testicular sperm maturation. An intergenerational contribution from paternal piRNAs has also been proposed. Together with insights into piRNAs in oocytes and early embryos in mice and other mammals, these newly proposed functions of mammalian piRNAs invite further investigations of piRNA dynamics during sperm maturation and fertilization as well as their roles in reproduction beyond gametogenesis.

DOI:
https://doi.org/10.1530/REP-22-0312
Volume 165: Issue 3,
Pages:
R91–R102
Restricted access

Placental leukocyte infiltration accompanies gestational changes induced by hyperthyroidism

María Belén Sánchez, Flavia Judith Neira, Tamara Moreno-Sosa, María Cecilia Michel Lara, Luciana Belén Viruel, María José Germanó, Elisa Olivia Pietrobon, Mariana Troncoso, Marta Soaje, Graciela Alma Jahn, Susana Ruth Valdez, and Juan Pablo Mackern-Oberti

In brief

The endocrine and immunological disruption induced by hyperthyroidism could alter gestation, placenta, and fetal development. This study suggests an immunological role of thyroid hormones in gestation.

Abstract

Thyroid dysfunctions lead to metabolic, angiogenic, and developmental alterations at the maternal–fetal interface that cause reproductive complications. Thyroid hormones (THs) act through their nuclear receptors that interact with other steroid hormone receptors. Currently, immunological regulation by thyroid status has been characterized to a far less extent. It is well known that THs exert regulatory function on immune cells and modulate cytokine expression, but how hyperthyroidism (hyper) modulates placental immunological aspects leading to placental alterations is unknown. This work aims to throw light on how hyper modulates immunological and morphological placental aspects. Control and hyper (induced by a daily s.c. injection of T4 0.25 mg/kg) Wistar rats were mated 8 days after starting T4 treatment and euthanized on days 19 (G19) and 20 (G20) of pregnancy. We removed the placenta to perform qPCR, flow cytometry, immunohistochemistry, Western blot and histological analysis, and amniotic fluid and serum to evaluate hormone levels. We observed that hyper increases the fetal number, fetal weight, and placental weight on G19. Moreover, hyper induced an endocrine imbalance with higher serum corticosterone and changed placental morphology, specifically the basal zone and decidua. These changes were accompanied by an increased mRNA expression of glucocorticoid receptor and monocyte chemoattractant protein-1, an increased mRNA and protein expression of prolactin receptor, and an increase in CD45+ infiltration. Finally, by in vitro assays, we evidenced that TH induced immune cell activation. In summary, we demonstrated that hyper modulates immunological and morphological placental aspects and induces fetal phenotypic changes, which could be related to preterm labor observed in hyper.

DOI:
https://doi.org/10.1530/REP-22-0210
Volume 165: Issue 3,
Pages:
235–248 (14 total)
Open access

Predicting the outcome of Thoroughbred stallion matings on the basis of dismount semen sample analyses

Robert John Aitken, Sarah Lambourne, and Ashlee Jade Medica

In brief

A capacity to predict the likelihood of pregnancy following natural matings would be of considerable benefit to the Thoroughbred horse breeding industry. In this article, we describe a strategy for achieving this outcome through the analysis of dismount samples, that achieved an overall accuracy of 94.6%.

Abstract

The purpose of this study was to determine whether the analysis of dismount semen samples from Thoroughbred stallions could be used to predict whether a given mating would result in a pregnancy. The analysis was based on 143 matings of 141 mares by a cohort of 7 Thoroughbred stallions over a 4-week period at an Australian Stud. The criteria of semen quality utilized in this analysis involved a preliminary assessment of the raw dismount sample in terms of semen volume, sperm number, and sperm movement characteristics using an iSperm® Equine portable device. Following this initial assessment, a subpopulation of progressively motile spermatozoa was isolated by virtue of the cells ability to migrate across a 5 µm polycarbonate filter in a Samson™ isolation chamber over a 15-minute period. These isolated cells were again evaluated for their number and quality of movement using the iSperm® system and, in addition, assessed for their ability to reduce WST-1, a membrane impermeant tetrazolium salt. These data were then combined with additional information describing the ages of the animals used in this study, their historical breeding records, and mating frequency during the breeding season. The total data set was then used to predict the occurrence of pregnancy, as confirmed by ultrasound at ~14 days post mating. The criteria used to predict fertility in the ensuing multivariate discriminant analysis were optimized for each individual stallion. Using this strategy, we were able to successfully predict the outcome of a cover with an overall accuracy of 94.6%.

DOI:
https://doi.org/10.1530/REP-22-0309
Volume 165: Issue 3,
Pages:
281–288 (8 total)
Restricted access

SMAD6 inhibits granulosa cell proliferation and follicle growth rate in carrier and noncarrier heifers of the Trio allele

Rafael R Domingues, Fabiana S Andrade, Joao Paulo N Andrade, Sadrollah M Moghbeli, Victor Gomez-Leon, Guilherme Madureira, Marco R B Mello, Brian W Kirkpatrick, and Milo C Wiltbank

In brief

Follicle selection is a key event in monovular species. In this manuscript, we demonstrate the role of SMAD6 in promoting decreased granulosa cell proliferation and follicle growth rate in carriers vs noncarriers of the Trio allele and after vs before follicle deviation.

Abstract

Cattle are generally considered a monovular species; however, recently, a bovine high fecundity allele, termed the Trio allele, was discovered. Carriers of Trio have an elevated ovulation rate (3–5), while half-sibling noncarriers are monovular. Carriers of the Trio allele have overexpression in granulosa cells of SMAD6, an inhibitor of oocyte-derived regulators of granulosa cell proliferation and differentiation. In experiment 1, follicle size was tracked for each follicle during a follicular wave. Follicle growth rate was greater before vs after follicle deviation in both carriers and noncarriers. Additionally, follicle growth rate was consistently less in carriers vs noncarriers. In experiment 2, we collected granulosa cells from follicles before and after deviation for evaluation of granulosa cell gene expression. Granulosa cell proliferation was less in carriers vs noncarriers and after vs before follicle deviation (decreased expression of cell cycle genes CCNB1 and CCNA2). The decreased granulosa cell proliferation in noncarriers after deviation was associated with increased SMAD6 expression. Similarly, in experiment 3, decreased expression of SMAD6 in granulosa cells of noncarriers cultured in vitro for 60 h was associated with increased expression of cell cycle genes. This suggests that SMAD6 may not just be inhibiting follicle growth rate in carriers of Trio but may also play a role in the decreased follicle growth after deviation in noncarriers. The hypotheses were supported that (1) follicle growth and granulosa cell proliferation decrease after deviation in both carriers and noncarriers and that (2) granulosa cell proliferation is reduced in carriers compared to noncarriers.

DOI:
https://doi.org/10.1530/REP-22-0232
Volume 165: Issue 3,
Pages:
269–279 (11 total)
Restricted access

Visfatin increases the invasive potential of ovarian granulosa tumor spheroids by reprogramming glucose metabolism

Justyna Gogola-Mruk, Weronika Marynowicz, Kinga Krawczyk, and Anna Ptak

Visfatin is an adipokine with nicotinamide phosphoribosyltransferase (NAMPT) activity, the concentration of which is higher in ascitic fluid than in serum, and is associated with ovarian cancer peritoneal dissemination. Furthermore, potentially important effects of visfatin on glucose metabolism have been previously reported. However, the mechanism underlying the effects of visfatin on ovarian cancer cell invasion, and whether this involves altered glucose metabolism, has not been elucidated. Here we tested the hypothesis that visfatin, which can reprogram cancer metabolism, promotes invasion by ovarian cancer spheroids. Visfatin increased glucose transporter (GLUT)1 expression and glucose uptake in adult granulosa cell tumor-derived spheroid cells (KGN), and also increased the activities of hexokinase 2 and lactate dehydrogenase. We showed a visfatin-induced increase in glycolysis in KGN cells. Moreover, visfatin increased the potential invasiveness of KGN spheroid cells by upregulating MMP2 (matrix metalloproteinase 2) and downregulating CLDN3 and CLDN4 (claudin 3 and 4) gene expression. Interestingly, an inhibitor of GLUT1 (STF-31) and lactate dehydrogenase (LDHA) abolished the stimulatory effect of visfatin on the potential invasiveness of KGN cells. More importantly, silencing expression of the NAMPT gene in KGN cells demonstrated its important effect on glycolysis and invasiveness in adult granulosa cell tumor cells. In summary, visfatin appears to increase adult granulosa cell tumor invasiveness through effects on glucose metabolism, and to be an important regulator of glucose metabolism in these cells

DOI:
https://doi.org/10.1530/REP-22-0443
Accepted Manuscripts
Restricted access

Whole genome DNA methylation analysis of the sperm in relation to bull fertility

Ying Zhang, Camila Bruna de Lima, Rémi Labrecque, and Marc-Andre Sirard

Subfertile bulls may cause huge economic losses in dairy production since their semen could be used to inseminate thousands of cows by artificial insemination. This study adopted whole-genome enzymatic methyl sequencing and aimed to identify candidate DNA methylation markers in bovine sperm that correlate with bull fertility. Twelve bulls were selected (High Bull Fertility = 6; Low Bull Fertility = 6) based on the industry’s internally used Bull Fertility Index (BFI). After sequencing, a total of 450 CpG had a DNA methylation difference higher than 20% (q<0.01) had been screened. The sixteen most significant differentially methylated regions(DMRs) were identified using a 10% methylation difference cut-off (q<5.88x10-16). Interestingly, most of the differentially methylated cytosines (DMCs) and DMRs were distributed on the X and Y chromosomes, demonstrating that the sex chromosomes play essential roles in bull fertility. Additionally, the functional classification showed that the beta-defensin family, zinc finger protein family, and olfactory and taste receptors could be clustered. Moreover, the enriched G protein-coupled receptors such as neurotransmitter receptors, taste receptors, the olfactory receptor family, and ion channels indicated that the acrosome reaction and capacitation processes are pivotal for bull fertility. In conclusion, this study identified the sperm-derived bull fertility-associated DMRs and DMCs at the whole genome level, which could complement and integrate into the existing genetic evaluation methods, increasing our decisive capacity to select good bulls and explain bull fertility better in the future.

DOI:
https://doi.org/10.1530/REP-22-0283
Accepted Manuscripts