ORIGINAL RESEARCH

steroidogenesis in porcine granulosa cell

The Role of Androgens in Ovarian Follicular Development: From Fertility to Ovarian Cancer

Androgens, steroid hormones produced by follicular cells, play a crucial role in the regulation of ovarian function. They affect folliculogenesis directly through androgen receptors (ARs) or indirectly through aromatization to estrogens. Androgens are thought to be primarily involved in preantral follicle growth and prevention of follicular atresia. It also seems possible that they are involved in the activation of primordial follicles. According to the World Health Organization, endocrine-disrupting chemicals (EDCs) are substances that alter hormonal signaling. EDCs comprise a wide variety of synthetic or natural chemicals arising from anthropogenic, industrial, agricultural, and domestic sources. EDCs interfere with natural regulation of the endocrine system by either mimicking or blocking the function of endogenous hormones as well as acting directly on gene expression or through epigenetic modifications. Disruptions in ovarian processes caused by EDCs may originate adverse outcomes such as anovulation, infertility, or premature ovarian failure. In this chapter, we aim to point out a possible involvement of androgen excess or deficiency in the regulation of ovarian function. We will summarize the effects of EDCs expressing antiandrogenic or androgenic activity on female physiology. Continuous exposition to even small concentration of such compounds can initiate oncogenesis within the ovary

The Primordial to Primary Follicle Transition — A Reliable Marker of Ovarian Function

In many mammalian species, including humans, folliculogenesis begins in fetal life and progresses throughout adulthood. The growing follicles progress from a reserve of primordial follicles that constitute the pool of female gametes for the entire life. Primordial follicles may begin to grow either immediately after forming or at clearly defined species-specific gap. Alternatively, some follicles may become quiescent before they either degenerate or resume growth several months or years afterwards. The rate of follicular assembly and the primordial to primary follicle transition is a critical step in female fertility. Therefore, disturbed coordination of the formation of primordial follicles and activation of their growth may entail some reproductive disorders. A poor initial reserve or the precocious primordial follicle depletion will result in infertility that, in women, is escorted by a shortened reproductive lifespan and early menopause. Therefore, it seems necessary to reach a profounder understanding of the molecular and cellular mechanisms controlling follicular development during preantral transition. In vitro growth of isolated immature ovarian follicles (IVGF) appears as an emerging technology, allowing to expand the fertility options in particular ovarian disorders or after cancer treatmen

Androgen receptor in early apoptotic follicles in the porcine ovary at pregnancy.

Localization of androgen receptor (AR) was investigated in ovarian follicles developing and undergoing atresia during pregnancy in the pig. Immunohistochemical staining was conducted on ovarian antral follicles isolated on different days of gestation: 10, 18, 32, 50, 70, and 90. Paraffin sections were also subjected to in situ DNA labeling. TUNEL staining revealed the presence of positive follicles on all days of pregnancy but the amount of atretic follicles increased with time. However, even on day 90 of gestation many follicles were normal, with no signs of atresia. In atretic follicles, apoptotic cells were localized predominantly in the granulosa while theca was much less affected. Atretic follicles with many apoptotic cells were negative for AR. Nuclear immunostaining for AR was positive in follicles with limited amount of apoptotic cells. The same relationship was observed in ovarian follicles isolated at various days of pregnancy

Analysis of localisation of megakaryocytes in the bone marrow of the rat : possible relationship with innervations

Piątek Małgorzata, Polaniak Renata, Tabarowski Zbigniew, Staśkiewicz Wiktoria, Kiciak Agata, Grajek Mateusz. Analysis of localisation of megakaryocytes in the bone marrow of the rat- possible relationship with innervations. Journal of Education, Health and Sport. 2022;12(7):479-492. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2022.12.07.049 https://apcz.umk.pl/JEHS/article/view/JEHS.2022.12.07.049 https://zenodo.org/record/6828868 The journal has had 40 points in Ministry of Education and Science of Poland parametric evaluation. Annex to the announcement of the Minister of Education and Science of December 21, 2021. No. The journal has had 40 points in Ministry of Education and Science of Poland parametric evaluation. Annex to the announcement of the Minister of Education and Science of December 21, 2021. No. 32343. Has a Journal's Unique Identifier: 201159. Scientific disciplines assigned: Physical Culture Sciences (Field of Medical sciences and health sciences); Health Sciences (Field of Medical Sciences and Health Sciences). Punkty Ministerialne z 2019 - aktualny rok 40 punktów. Załącznik do komunikatu Ministra Edukacji i Nauki z dnia 21 grudnia 2021 r. Lp. 32343. Posiada Unikatowy Identyfikator Czasopisma: 201159. Przypisane dyscypliny naukowe: Nauki o kulturze fizycznej (Dziedzina nauk medycznych i nauk o zdrowiu); Nauki o zdrowiu (Dziedzina nauk medycznych i nauk o zdrowiu). © The Authors 2022; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 22.06.2022. Revised: 22.06.2022. Accepted: 13.07.2022. ANALYSIS OF LOCALISATION OF MEGAKARYOCYTES IN THE BONE MARROW OF THE RAT- POSSIBLE RELATIONSHIP WITH INNERVATIONS Małgorzata Piątek1, Renata Polaniak1*, Zbigniew Tabarowski2, Wiktoria Staśkiewicz3, Agata Kiciak3, Mateusz Grajek4 1Department of Human Nutrition, Department of Dietetics, Faculty of Health Sciences in Bytom, Medical University of Silesia in Katowice, Poland 2Department of Experimental Hematology, Department of Animal Physiology, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Poland 3Department of Food Technology and Quality Evaluation, Department of Dietetics, Faculty of Health Sciences in Bytom, Medical University of Silesia in Katowice, Poland 4Department of Public Health, Department of Public Health Policy, Faculty of Health Sciences in Bytom, Medical University of Silesia in Katowice, Poland *Correspondence: Renata Polaniak, PhD; Jordana 19, 41808 Zabrze (Poland); [email protected] ABSTRACT Within the bone marrow, megakaryocytes thrive in an environment rich in numerous growth factors that influences their development, maturation, and may play a role in signaling the start of platelet production. Up to now, experiments concerning localisation of various populations of cells in rat bone marrow showed their certain relationship to the blood vessels. There are strikingly few reliable sources of information available in the literature that confirm the relationship between the localisation of these cells in connection to nerve fibers. Therefore the purpose of the present study was to examine if megakaryocytes are located in a defined relation to sensory and sympathetic nerve fibers. The study used immature 6 weeks rats of the Wistar breed of both sexes. The animals were kept under constant lighting conditions (12 hours light-dark cycle) and temperature, and had unrestricted access to water and food (standard laboratory rodent feed).Double immunostaining method was applied with usage of secondary antibodies conjugated with fluorochromes (Cy3 and DTAF) to identify cells. The antibody against CD42d was used to immunolocalise megakaryocytes, while in order to identify the distribution of nerves, antibodies anti-NPY (for detection sympathetic nerve fibers) and anti-PGP 9.5 (sensory nerve fibers) were applied. These findings showed the presence of megakaryocyte and megakaryoblastic cells which were distributed with a close relationship to the blood vessels but some of them were located in parenchyma. It was shown that NPY-positive and PGP 9.5-positive cells were present in the vicinity of blood cells. Furthermore, some megakaryocytes were located near PGP 9.5-labelled cells. The relationship between sympathetic nerve fibers containing neuropeptide Y and megakaryocytes was also detected. The presence of megakaryocytes with a close vicinity of nerve fibers suggests the influence of the released neurotransmitters on the location of the megakaryocytes in the bone marrow. KEYWORDS: megakaryocytes, platelets, bone marrow, innervation, neuropeptide Y, PGP 9.

The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles

We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6-8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10(-7) M), 2-Hf (1.7 x 10(-4) M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3 beta-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3 beta-HSD and P450c17 increase on mRNA level, but decreased their protein expression. This was against our expectations but the reason for that finding remains undiscovered, intriguing and worth reporting. These results suggest that alike, steroidogenic enzymes activity and their expression is associated with the presence of androgens and AR in the porcine ovary

Role of Calcitonin Gene-Related Peptide in Bone Repair after Cyclic Fatigue Loading

Calcitonin gene related peptide (CGRP) is a neuropeptide that is abundant in the sensory neurons which innervate bone. The effects of CGRP on isolated bone cells have been widely studied, and CGRP is currently considered to be an osteoanabolic peptide that has effects on both osteoclasts and osteoblasts. However, relatively little is known about the physiological role of CGRP in-vivo in the skeletal responses to bone loading, particularly fatigue loading.We used the rat ulna end-loading model to induce fatigue damage in the ulna unilaterally during cyclic loading. We postulated that CGRP would influence skeletal responses to cyclic fatigue loading. Rats were fatigue loaded and groups of rats were infused systemically with 0.9% saline, CGRP, or the receptor antagonist, CGRP(8-37), for a 10 day study period. Ten days after fatigue loading, bone and serum CGRP concentrations, serum tartrate-resistant acid phosphatase 5b (TRAP5b) concentrations, and fatigue-induced skeletal responses were quantified. We found that cyclic fatigue loading led to increased CGRP concentrations in both loaded and contralateral ulnae. Administration of CGRP(8-37) was associated with increased targeted remodeling in the fatigue-loaded ulna. Administration of CGRP or CGRP(8-37) both increased reparative bone formation over the study period. Plasma concentration of TRAP5b was not significantly influenced by either CGRP or CGRP(8-37) administration.CGRP signaling modulates targeted remodeling of microdamage and reparative new bone formation after bone fatigue, and may be part of a neuronal signaling pathway which has regulatory effects on load-induced repair responses within the skeleton

Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell–Related Genes

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs