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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
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

dmrtb1 is involved in the testicular development in Larimichthys crocea

Yan Feng, Zhaowei Zhong, Haifu Wan, Ziping Zhang, Pengfei Zou, Peng Lin, Yonghua Jiang, and Yilei Wang

In brief

dmrtb1 performs critical functions in sex determination/differentiation and gonadal development in many organisms, but its role in teleost is rarely studied. Through gene cloning, in situ hybridization, and RNA interference technology, the function of dmrtb1 in testicular development of large yellow croaker (Larimichthys crocea) was studied; our study will be helpful in understanding further the molecular regulation mechanism of Lcdmrtb1/Lcdmrt6 in testicular development in L. crocea, and our results enrich the theory of fish dmrts involved in reproductive regulation and provide a new idea for sex control breeding of L. crocea by manipulating reproductive pathway.

Abstract

Doublesex- and mab-3-related transcription factor B1 (dmrtb1/dmrt6) belongs to one of the members of DMRT family, which performs critical functions in sex determination and differentiation, gonadal development, and functional maintenance. However, knowledge of its exact mechanism remains unclear in teleost. Very little is known about the role of dmrtb1 in the gonad development of Larimichthys crocea. In this study, a dmrtb1 homolog in L. crocea named as Lcdmrtb1 with the full-length cDNA was isolated and characterized. Except for the conserved DM domain, the other regions had low homology. Of the tissues sampled, Lcdmrtb1 was only found to be highly expressed in the testis. In situ hybridization of testis revealed Lcdmrtb1 in both spermatogonia and spermatocytes. After Lcdmrtb1 interference in the testis cells (LYCT) of L. crocea, the expression levels of Lcdmrtb1 and Lcdmrt1 were significantly decreased; subsequently, testicular cell morphology changed from fibrous to round and their growth rate slowed. Similarly, the expression levels of Lcdmrtb1, Lcdmrt1, sox9a/b, and amh were significantly decreased after RNAi in the testis. Furthermore, it was discovered that the spermatogonia had disappeared, and the Sertoli cells had been reduced. The results of immunohistochemistry showed that the expression of Sox9 protein in the testis was not detected after dmrtb1 was knocked down. These results indicated that the absence of Lcdmrtb1 not only greatly inhibited cell growth and destroyed the morphology of testis cells but also down-regulated Lcdmrt1 expression in the testis. This study will be helpful in understanding further the molecular regulation mechanism of Lcdmrtb1/Lcdmrt6 in testicular development in L. crocea.

DOI:
https://doi.org/10.1530/REP-22-0214
Volume 165: Issue 2,
Pages:
159–170 (12 total)
Free access

Dynamics of gametes and embryos in the oviduct: what can in vivo imaging reveal?

Shang Wang and Irina V Larina

In brief

In vivo imaging of gametes and embryos in the oviduct enables new studies of the native processes that lead to fertilization and pregnancy. This review article discusses recent advancements in the in vivo imaging methods and insights which contribute to understanding the oviductal function.

Abstract

Understanding the physiological dynamics of gametes and embryos in the fallopian tube (oviduct) has significant implications for managing reproductive disorders and improving assisted reproductive technologies. Recent advancements in imaging of the mouse oviduct in vivo uncovered fascinating dynamics of gametes and embryos in their native states. These new imaging approaches and observations are bringing exciting momentum to uncover the otherwise-hidden processes orchestrating fertilization and pregnancy. For mechanistic investigations, in vivo imaging in genetic mouse models enables dynamic phenotyping of gene functions in the reproductive process. Here, we review these imaging methods, discuss insights recently revealed by in vivo imaging, and comment on emerging directions, aiming to stimulate new in vivo studies of reproductive dynamics.

DOI:
https://doi.org/10.1530/REP-22-0250
Volume 165: Issue 2,
Pages:
R25–R37
Restricted access

Endoplasmic reticulum stress promotes endometrial fibrosis through the TGF-β/SMAD pathway

Meng Bao, Qiwen Feng, Liping Zou, Jin Huang, Changhong Zhu, and Wei Xia

In brief

Intrauterine adhesion (IUA) is one of the main causes of female infertility. This study reveals that endoplasmic reticulum stress activation upregulates the TGF-β/SMAD pathway to induce epithelial–mesenchymal transition and promote endometrial fibrosis in an IUA model.

Abstract

IUA is a common gynecological disease and is a leading cause of female infertility. Mechanical or infectious damage to the endometrial basal layer can lead to endometrial fibrosis, which is the most common cause of IUA. Endoplasmic reticulum stress (ERS), the transforming growth factor beta signaling pathway (TGF-β/SMAD) and epithelial–mesenchymal transition (EMT) are important factors promoting endometrial fibrosis. The purpose of this study was to determine the up- and downstream regulatory relationships of the above three in the process of endometrial fibrosis. The rat IUA model was induced by double injury method and prophylactic injection of the ERS inhibitor 4-phenylbutyric acid (4-PBA) was given in vivo. The ERS activator tunicamycin and the TGF-β/SMAD pathway inhibitor A 83-01 were used in human endometrial epithelial cells (HEECs) in vitro. Masson’s trichrome, Sirius red staining, immunohistochemistry, immunofluorescence and Western blot analyses were used to determine ERS, TGF-β/SMAD pathway, EMT and fibrosis markers in the uterine tissue and HEECs of the different treatment groups. In animal experiments, ERS and the TGF-β/SMAD pathway had been activated and EMT occurred in an in vivo model of IUA but was suppressed in animals treated with prophylactic 4-PBA. In in vitro experiments, tunicamycin-treated HEECs had increased the activation of ERS, the abundance of TGF-β/SMAD pathway and fibrosis markers while EMT occurred, but the TGF-β/SMAD pathway and EMT were significantly inhibited in the tunicamycin+A 83-01 group. Our data suggest that increased ERS can induce EMT and promote endometrial fibrosis through the TGF-β/SMAD pathway.

DOI:
https://doi.org/10.1530/REP-22-0294
Volume 165: Issue 2,
Pages:
171–182 (12 total)
Free access

Immunomodulation for unexplained recurrent implantation failure: where are we now?

Geneviève Genest, Shorooq Banjar, Walaa Almasri, Coralie Beauchamp, Joanne Benoit, William Buckett, Frederick Dzineku, Phil Gold, Michael H Dahan, Wael Jamal, Isaac Jacques Kadoch, Einav Kadour-Peero, Louise Lapensée, Pierre Miron, Talya Shaulov, Camille Sylvestre, Togas Tulandi, Bruce D Mazer, Carl A Laskin, and Neal Mahutte

In brief

Immune dysfunction may contribute to or cause recurrent implantation failure. This article summarizes normal and pathologic immune responses at implantation and critically appraises currently used immunomodulatory therapies.

Abstract

Recurrent implantation failure (RIF) may be defined as the absence of pregnancy despite the transfer of ≥3 good-quality blastocysts and is unexplained in up to 50% of cases. There are currently no effective treatments for patients with unexplained RIF. Since the maternal immune system is intricately involved in mediating endometrial receptivity and embryo implantation, both insufficient and excessive endometrial inflammatory responses during the window of implantation are proposed to lead to implantation failure. Recent strategies to improve conception rates in RIF patients have focused on modulating maternal immune responses at implantation, through either promoting or suppressing inflammation. Unfortunately, there are no validated, readily available diagnostic tests to confirm immune-mediated RIF. As such, immune therapies are often started empirically without robust evidence as to their efficacy. Like other chronic diseases, patient selection for immunomodulatory therapy is crucial, and personalized medicine for RIF patients is emerging. As the literature on the subject is heterogenous and rapidly evolving, we aim to summarize the potential efficacy, mechanisms of actions and side effects of select therapies for the practicing clinician.

DOI:
https://doi.org/10.1530/REP-22-0150
Volume 165: Issue 2,
Pages:
R39–R60
Restricted access

m6A methyltransferase METTL3 inhibits endometriosis by regulating alternative splicing of MIR17HG

Qian Li, Li Yang, Feng Zhang, Jiaxi Liu, Min Jiang, Yannan Chen, and Chenchen Ren

In brief

Inflammation and abnormal immune response are the key processes in the development of endometriosis (EMs), and m6A modification can regulate the inflammatory response. This study reveals that METTL3-mediated N6-methyladenosine (m6A) modification plays an important role in EMs.

Abstract

m6A modification is largely involved in the development of different diseases. This study intended to investigate the implication of m6A methylation transferase methyltransferase like 3 (METTL3) in EMs. EMs- and m6A-related mRNAs and long non-coding RNAs were identified through bioinformatics analysis. Next, EM mouse models established by endometrial autotransplantation and mouse endometrial stromal cell (mESC) were prepared and treated with oe-METTL3 or sh-MIR17HG for pinpointing the in vitro and in vivo effects of METTL3 on EMs in relation to MIR17HG through the determination of mESC biological processes as well as estradiol (E2) and related lipoprotein levels. We demonstrated that METTL3 and MIR17HG were downregulated in the EMs mouse model. Overexpression of METTL3 suppressed the proliferation, migration, and invasion of mESCs. In addition, METTL3 enhanced the expression of MIR17HG through m6A modification. Moreover, METTL3 could inhibit the E2 level and alter related lipoprotein levels in EMs mice through the upregulation of MIR17HG. The present study highlighted that the m6A methylation transferase METTL3 prevents EMs progression by upregulating MIR17HG expression.

DOI:
https://doi.org/10.1530/REP-22-0102
Volume 165: Issue 2,
Pages:
197–208 (12 total)