Immunohistochemical localization of integrin alpha V beta 3 and osteopontin suggests that they do not interact during embryo implantation in ruminants

BACKGROUND: It has been suggested that trophoblast attachment requires co-expression of integrin alpha V beta 3 and its ligand osteopontin at the fetal-maternal interface. Until now the expression patterns of integrin alpha V beta 3 and osteopontin in the pregnant bovine uterus were unknown. The objectives of this study were to localize integrin alpha V beta 3 and osteopontin in bovine and sheep endometrium during the periimplantation period and to compare the distribution patterns using antibodies that had not yet been tested in sheep. METHODS: Cell compartments within endometrial tissue sections were scored for immunohistochemical staining intensity and data were analyzed to determine the effects of day of pregnancy or cycle. RESULTS: In pregnant bovine endometrium, integrin alpha V beta 3 was detected in luminal epithelium, stroma, myometrium and smooth muscle. A strong band of immunoreactivity was observed in the subepithelial stroma of intercaruncular regions, but there was reduced reactivity in the caruncles and glands. Bovine trophoblast did not express integrin alpha V beta 3 at any stage of pregnancy. In ovine endometrium a different pattern of staining for integrin alpha V beta 3 was observed. Reactivity was not present in the luminal epithelium or trophoblast. There was strong staining of the deep glands and no reactivity in the superficial glands. Osteopontin distribution was similar for sheep and cattle. For both species, apical staining was present on the luminal epithelium and glands and on embryonic tissues. CONCLUSION: In ruminants, integrin alpha V beta 3 and osteopontin do not co-localize at the fetal-maternal interface indicating that these proteins could not interact to facilitate embryo attachment as has been proposed in other species

The effects of estrogen, its antagonist ICI 182, 780, and interferon-tau on the expression of estrogen receptors and integrin alphaV beta 3 on cycle day 16 in bovine endometrium

We have shown previously that downregulation of intercaruncular stromal integrin α(v)β(3 )in bovine endometrium on day 16 of the estrous cycle coincided with the antibody recognition of estrogen receptors (ER) in the luminal epithelium. In pregnancy, these changes were not observed. Our hypothesis was that on day 16 of the estrous cycle, estrogen from the dominant follicle causes a reduction in integrin α(v)β(3 )and affects ERα in the luminal epithelium. The pregnancy recognition protein, interferon-τ (IFN-τ), may prevent downregulation of integrin α(v)β(3 )and suppress ERα expression in the luminal epithelium. On days 14 to 16, heifers received uterine infusions of the anti-estrogen ICI 182, 780, estradiol 17β, IFN-τ or the saline control. On day 16, reproductive tracts were collected for analysis of integrin α(v)β(3 )and ERα. Estrogen receptor α immunoreactivity was largely restricted to the luminal epithelium in control animals. Using anti-ERα recognizing the amino terminus, estrogen-treated animals showed reactivity in the stroma, shallow and deep glands and myometrium as is typical of estrus, whereas ICI 182, 870 treated heifers showed little or no reactivity. In contrast, carboxyl terminus-directed antibodies showed a widespread distribution of ERα with reactivity detected in the uterine epithelium, stroma and myometrium of both estrogen and ICI 182, 780 treated animals. Heifers treated with IFN-τ had low ERα reactivity overall. Control and IFN-τ treated heifers had lower intercaruncular stromal expression of integrin α(v)β(3 )in comparison to estrogen and ICI 182, 780 treatments. Overall, the results suggest that on day 16 of the estrous cycle, estrogen effects on integrin α(v)β(3 )are indirect and do not directly involve ERα in the luminal epithelium. During pregnancy, interferon-tau may block ERα in the luminal epithelium but likely does not rescue integrin α(v)β(3 )expression

Current global status of male reproductive health

BACKGROUND: The widespread interest in male reproductive health (MRH), fueled by emerging evidence, such as the global decline in sperm counts, has intensified concerns about the status of MRH. Consequently, there is a pressing requirement for a strategic, systematic approach to identifying critical questions, collecting pertinent information, and utilizing these data to develop evidence-based strategies. The methods for addressing these questions and the pathways toward their answers will inevitably vary based on the variations in cultural, geopolitical, and health-related contexts. To address these issues, a conjoint ESHRE and Male Reproductive Health Initiative (MRHI) Campus workshop was convened.OBJECTIVE AND RATIONALE: The three objectives were: first, to assess the current state of MRH around the world; second, to identify some of the key gaps in knowledge; and, third, to examine how MRH stakeholders can collaboratively generate intelligent and effective paths forward.SEARCH METHODS: Each expert reviewed and summarized the current literature that was subsequently used to provide a comprehensive overview of challenges related to MRH.OUTCOMES: This narrative report is an overview of the data, opinions, and arguments presented during the workshop. A number of outcomes are presented and can be summarized by the following overarching themes: MRH is a serious global issue and there is a plethora of gaps in our understanding; there is a need for widespread international collaborative networks to undertake multidisciplinary research into fundamental issues, such as lifestyle/environmental exposure studies, and high-quality clinical trials; and there is an urgent requirement for effective strategies to educate young people and the general public to safeguard and improve MRH across diverse population demographics and resources.LIMITATIONS REASONS FOR CAUTION: This was a workshop where worldwide leading experts from a wide range of disciplines presented and discussed the evidence regarding challenges related to MRH. While each expert summarized the current literature and placed it in context, the data in a number of areas are limited and/or sparse. Equally, important areas for consideration may have been missed. Moreover, there are clear gaps in our knowledge base, which makes some conclusions necessarily speculative and warranting of further study.WIDER IMPLICATIONS: Poor MRH is a global issue that suffers from low awareness among the public, patients, and heathcare professionals. Addressing this will require a coordinated multidisciplinary approach. Addressing the significant number of knowledge gaps will require policy makers prioritizing MRH and its funding.STUDY FUNDING/COMPETING INTERESTS: The authors would like to extend their gratitude to ESHRE for providing financial support for the Budapest Campus Workshop, as well as to Microptic S.L. (Barcelona) for kindly sponsoring the workshop. P.B. is the Director of the not-for-profit organization Global Action on Men's Health and receives fees and expenses for his work, (which includes the preparation of this manuscript). Conflicts of interest: C.J.D.J., C.L.R.B., R.A.A., P.B., M.P.C., M.L.E., N.G., N.J., C.K., AAP, M.K.O., S.R.-H., M.H.V.-L.: ESHRE Campus Workshop 2022 (Travel support-personal). C.J.D.J.: Cambridge University Press (book royalties-personal). ESHRE Annual Meeting 2022 and Yale University Panel Meeting 2023 (Travel support-personal). C.L.R.B.: Ferring and IBSA (Lecture), RBMO editor (Honorarium to support travel, etc.), ExSeed and ExScentia (University of Dundee), Bill &amp; Melinda Gates Foundation (for research on contraception). M.P.C.: Previously received funding from pharmaceutical companies for health economic research. The funding was not in relation to this work and had no bearing on the contents of this work. No funding from other sources has been provided in relation to this work (funding was provided to his company Global Market Access Solutions). M.L.E.: Advisor to Ro, Doveras, Next, Hannah, Sandstone. C.K.: European Academy of Andrology (Past president UNPAID), S.K.: CEO of His Turn, a male fertility Diagnostic and Therapeutic company (No payments or profits to date). R.I.M.: www.healthymale.org.au (Australian Government funded not for profit in men's health sector (Employed as Medical Director 0.2 FET), Monash IVF Pty Ltd (Equity holder)). N.J.: Merck (consulting fees), Gedeon Richter (honoraria). S.R.-H.: ESHRE (Travel reimbursements). C.N.: LLC (Nursing educator); COMMIT (Core Outcomes Measures for Infertility Trials) Advisor, meeting attendee, and co-author; COMMA (Core Outcomes in Menopause) Meeting attendee, and co-author; International Federation of Gynecology and Obstetrics (FIGO) Delegate Letters and Sciences; ReproNovo, Advisory board; American Board of Urology Examiner; American Urological Association Journal subsection editor, committee member, guidelines co-author Ferring Scientific trial NexHand Chief Technology Officer, stock ownership Posterity Health Board member, stock ownership. A.P.: Economic and Social Research Council (A collaborator on research grant number ES/W001381/1). Member of an advisory committee for Merck Serono (November 2022), Member of an advisory board for Exceed Health, Speaker fees for educational events organized by Mealis Group; Chairman of the Cryos External Scientific Advisory Committee: All fees associated with this are paid to his former employer The University of Sheffield. Trustee of the Progress Educational Trust (Unpaid). M.K.O.: National Health and Medical Research Council and Australian Research Council (Funding for research of the topic of male fertility), Bill and Melinda Gates Foundation (Funding aimed at the development of male gamete-based contraception), Medical Research Future Fund (Funding aimed at defining the long-term consequences of male infertility). M.H.V.-L.: Department of Sexual and Reproductive Health and Research (SRH)/Human Reproduction Programme (HRP) Research Project Panel RP2/WHO Review Member; MRHI (Core Group Member), COMMIT (member), EGOI (Member); Human Reproduction (Associate Editor), Fertility and Sterility (Editor), AndroLATAM (Founder and Coordinator).</p

Epigenetic Mechanisms Regulate Stem Cell Expressed Genes Pou5f1 and Gfra1 in a Male Germ Cell Line

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia – here, predominantly mediated by HDAC-containing protein complexes

Testis-specific transcription mechanisms promoting male germ-cell differentiation

Male germ-cell differentiation requires spermatogenic stage- and cell-specific gene expression that is achieved by unique chromatin remodeling, transcriptional control and the expression of testis-specific genes or isoforms. Recent findings have shown that the testis has specialized transcription complexes that coordinate the differentiation program of spermatogenesis. There are male germ cell-specific differences in the components of the general transcription machinery. These include upregulated expression of the TATA-binding protein (TBP) family and its associated cofactors. Importantly, a member of the TBP family, TBP-like factor (TLF), has a distribution pattern that is dependent on the spermatogenic cycle and is essential for spermatogenesis. Interestingly TBP-associated factor (TAF7), a factor of the transcription factor (TF)IID complex, is exchanged at a critical stage in germ cell development for the testis-specific paralogue TAF7L. A compelling amount of data has established that cAMP-response-element modulator (CREM), a transcription factor responsive to the cAMP signal transduction pathway, drives expression of key testis-specific genes. In this review we summarize recent advances in the transcription machinery that is testis-specific, gene-selective and necessary for the process of spermatogenesis

ChIP-seq protocol for sperm cells and embryos to assess environmental impacts and epigenetic inheritance

Summary: In the field of epigenetic inheritance, delineating molecular mechanisms implicated in the transfer of paternal environmental conditions to descendants has been elusive. This protocol details how to track sperm chromatin intergenerationally. We describe mouse model design to probe chromatin states in single mouse sperm and techniques to assess pre-implantation embryo chromatin and gene expression. We place emphasis on how to obtain high-quality and quantifiable data sets in sperm and embryos, as well as highlight the limitations of working with low input.For complete details on the use and execution of this protocol, please refer to Lismer et al. (2021)