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A field guide to Aurora kinase inhibitors: an oocyte perspective

Cecilia S Blengini, Gyu Ik Jung, Mansour Aboelenain, and Karen Schindler

In brief

The Aurora protein kinases have critical functions in controlling oocyte meiotic maturation. In this study, we describe an assay for examining their activation state in oocytes and establish the best working doses of three commonly used inhibitors.

Abstract

Several small molecule inhibitors exist for targeting Aurora kinase proteins in somatic cells. From this point of view, we evaluate the specificity of these inhibitors in mouse oocytes, and we demonstrate that MLN 8237 and AZD 1152 are specific for Aurora kinase A and Aurora kinase C, respectively, only when used at low concentrations.

DOI:
https://doi.org/10.1530/REP-22-0292
Volume 164: Issue 4,
Pages:
V5–V7
Restricted access

Hyperpolarization induces cytosolic alkalization of mouse sperm flagellum probably through sperm Na+/H+ exchanger

Sandra Hernández-Garduño, Julio C Chávez, Arturo Matamoros-Volante, Yoloxochitl Sánchez-Guevara, Paulina Torres, Claudia L Treviño, and Takuya Nishigaki

In brief

Hyperpolarization of the membrane potential is a crucial step for mammalian sperm maturation. This work demonstrates that this membrane potential change likely activates a sperm-specific sodium/proton exchanger to induce alkalization in mouse sperm flagellum.

Abstract

The sperm-specific sodium/proton exchanger (sNHE) is an indispensable protein for male fertility in mammals. Nevertheless, it is still unknown how mammalian sNHE is regulated. Evidence obtained from sea urchin sNHE indicates that hyperpolarization of plasma membrane potential (Vm), which is a hallmark of mammalian capacitation, positively regulates the sNHE. Therefore, we explored the activity of sNHE in mouse and human sperm by fluorescence imaging of intracellular pH (pHi) with a ratiometric dye, SNARF-5F. A valinomycin-induced Vm hyperpolarization elevated sperm flagellar pHi of WT mouse but not in sNHE-KO mouse. Moreover, this pHi increase was inhibited in a high K+ (40 mM) medium. These results support the idea that mouse sNHE is activated by Vm hyperpolarization. Interestingly, we observed different types of kinetics derived from valinomycin-induced alkalization, including some (30%) without any pHi changes. Our quantitative pHi determinations revealed that unresponsive cells had a high resting pHi (>7.5), suggesting that the activity of mouse sNHE is regulated by the resting pHi. On the other hand, valinomycin did not increase the pHi of human sperm in the head or the flagellum, regardless of their resting pHi values. Our findings suggest that the regulatory mechanisms of mammalian sNHEs are probably distinct depending on the species.

DOI:
https://doi.org/10.1530/REP-22-0101
Volume 164: Issue 4,
Pages:
125–134 (10 total)
Restricted access

Pregnancy preparation: redistribution of CCR7-positive cells in the rat uterus

Hannah Thomas, Erick McCloskey, and Virginia Rider

In brief

Changes in the endometrium prior to implantation may be critical in predicting pregnancy outcomes. This study shows that the endocrine system directs positional changes in CCR7+ cells before implantation, which may be critical for developing maternal tolerance.

Abstract

Suppression of the maternal immune system is vital for the implantation of the semi-allogeneic embryo. Although progress in understanding the dialogue between mother and embryo has been made, key interactions between maternal immune cells, hormones, and chemokines remain elusive. Uterine expression of the C-C chemokine receptor type 7 (CCR7) could recruit T regulatory cells and facilitate localized immune suppression. To test this concept, Ccr7 mRNA and protein were assessed in uterine tissue. Ccr7 mRNA expression peaked at day 4 in pregnant rat uteri and then declined at days 5 and 6. CCR7 protein showed similar quantitative changes. To test if female sex steroids affected the spatial distribution of CCR7-expressing cells, uteri from ovariectomized rats, progesterone-pretreated rats (2 mg daily), and progesterone-pretreated rats injected with estradiol (0.2 µg) were analyzed. Progesterone increased CCR7-positive (+) cells in the antimesometrial stroma. Progesterone and estradiol increased CCR7+ cells in the mesometrial stroma. Estradiol increased the density of cluster of differentiation 4 (CD4) positive cells in the mesometrial stromal region over progesterone alone. The density of cells expressing the T regulatory cell marker, forkhead box protein 3 (FOXP3), increased in the antimesometrial stroma in response to progesterone alone. Progesterone and estradiol increased FOXP3+ cells in the antimesometrial region of the stroma. Co-localization of CCR7, CD4, and FOXP3 in the stroma suggests CCR7+ cells are T regulatory cells. Polarization of CCR7+ cells in the endometrial stroma was an intrinsic response regulated by sex steroids and did not require the presence of an embryo.

DOI:
https://doi.org/10.1530/REP-22-0074
Volume 164: Issue 4,
Pages:
183–193 (11 total)
Restricted access

Production of extracellular vesicles from equine embryo-derived mesenchymal stromal cells

Zoe Tasma, Weilin Hou, Tanvi Damani, Kathleen Seddon, Matthew Kang, Yi Ge, David Hanlon, Fiona Hollinshead, Colin L Hisey, and Lawrence W Chamley

In brief

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promise as off-the-shelf therapeutics; however, producing them in sufficient quantities can be challenging. In this study, MSCs were isolated from preimplantation equine embryos and used to produce EVs in two commercially available bioreactor designs.

Abstract

Mesenchymal stromal cells (MSC) have recently been explored for their potential use as therapeutics in human and veterinary medicine applications, such as the treatment of endometrial inflammation and infertility. Allogeneic MSC-derived extracellular vesicles (EVs) may also provide therapeutic benefits with advantage of being an ‘off-the-shelf’ solution, provided they can be produced in large enough quantities, without contamination from bovine EVs contained in fetal bovine serum that is a common component of cell culture media. Toward this aim, we demonstrated the successful isolation and characterization of equine MSCs from preimplantation embryos. We also demonstrate that many of these lines can be propagated long-term in culture while retaining their differentiation potential and conducted a head-to-head comparison of two bioreactor systems for scalable EV production including in serum-free conditions. Based on our findings, the CELLine AD 1000 flasks enabled higher cell density cultures and significantly more EV production than the FiberCell system or conventional culture flasks. These findings will enable future isolation of equine MSCs and the scalable culture of their EVs for a wide range of applications in this rapidly growing field.

DOI:
https://doi.org/10.1530/REP-22-0215
Volume 164: Issue 4,
Pages:
143–154 (12 total)
Open access

RAC1 is involved in uterine myometrium contraction in the inflammation-associated preterm birth

Min Diao, Jin Zhou, Yunkai Tao, Zhaoyang Hu, and Xuemei Lin

In brief

Various etiologies can cause uterine myometrium contraction, which leads to preterm birth. This study demonstrates a new functional relationship between the Ras-related C3 botulinum toxin substrate 1 (RAC1) and uterine myometrium contraction in preterm birth.

Abstract

Preterm birth (PTB) is a public health issue. The World Health Organization has recommended the use of tocolytic treatment to inhibit preterm labour and improve pregnancy outcomes. Intrauterine inflammation is associated with preterm birth. RAC1 can modulate inflammation in different experimental settings. In the current study, we explored whether RAC1 can modulate spontaneous uterine myometrium contraction in a mouse model of lipopolysaccharide (LPS)-induced intrauterine inflammation. Subsequently, we recorded uterine myometrium contraction and examined uterine Rac1 expression in a mouse model of preterm birth and a case in pregnant women by Western blotting analysis. We also measured progesterone levels in the blood serum of mice. Murine myometrium was obtained 12 h post LPS treatment. Human myometrium was obtained at the time of caesarean section. We found that in the LPS-treated group of mice, uterine myometrium contraction was enhanced, protein levels and activation of RAC1 were increased and serum progesterone levels were decreased. The protein levels of RAC1 were also increased in preterm birth and in pregnant women. NSC23766, a RAC1 inhibitor, attenuated uterine myometrium contraction and diminished RAC1 activation and COX-2 expression. Furthermore, silencing of RAC1 suppressed cell contraction and COX-2 expression in vitro. In conclusion, our results suggested that RAC1 may play an important role in modulating uterine myometrium contraction. Consequently, intervening with RAC1 represents a novel strategy for the treatment of preterm birth.

DOI:
https://doi.org/10.1530/REP-21-0186
Volume 164: Issue 4,
Pages:
169–181 (13 total)
Free access

The role of angiotensin II and relaxin in vascular adaptation to pregnancy

Thu Ngoc Anh Doan, Tina Bianco-Miotto, Laura Parry, and Marnie Winter

In brief

There is a pregnancy-induced vasodilation of blood vessels, which is known to have a protective effect on cardiovascular function and can be maintained postpartum. This review outlines the cardiovascular changes that occur in a healthy human and rodent pregnancy, as well as different pathways that are activated by angiotensin II and relaxin that result in blood vessel dilation.

Abstract

During pregnancy, systemic and uteroplacental blood flow increase to ensure an adequate blood supply that carries oxygen and nutrients from the mother to the fetus. This results in changes to the function of the maternal cardiovascular system. There is also a pregnancy-induced vasodilation of blood vessels, which is known to have a protective effect on cardiovascular health/function. Additionally, there is evidence that the effects of maternal vascular vasodilation are maintained post-partum, which may reduce the risk of developing high blood pressure in the next pregnancy and reduce cardiovascular risk later in life. At both non-pregnant and pregnant stages, vascular endothelial cells produce a number of vasodilators and vasoconstrictors, which transduce signals to the contractile vascular smooth muscle cells to control the dilation and constriction of blood vessels. These vascular cells are also targets of other vasoactive factors, including angiotensin II (Ang II) and relaxin. The binding of Ang II to its receptors activates different pathways to regulate the blood vessel vasoconstriction/vasodilation, and relaxin can interact with some of these pathways to induce vasodilation. Based on the available literature, this review outlines the cardiovascular changes that occur in a healthy human pregnancy, supplemented by studies in rodents. A specific focus is placed on vasodilation of blood vessels during pregnancy; the role of endothelial cells and endothelium-derived vasodilators will also be discussed. Additionally, different pathways that are activated by Ang II and relaxin that result in blood vessel dilation will also be reviewed.

DOI:
https://doi.org/10.1530/REP-21-0428
Volume 164: Issue 4,
Pages:
R87–R99
Free access

Trophectoderm formation: regulation of morphogenesis and gene expressions by RHO, ROCK, cell polarity, and HIPPO signaling

Vernadeth B Alarcon and Yusuke Marikawa

In brief

Trophectoderm is the first tissue to differentiate in the early mammalian embryo and is essential for hatching, implantation, and placentation. This review article discusses the roles of Ras homolog family members (RHO) and RHO-associated coiled-coil containing protein kinases (ROCK) in the molecular and cellular regulation of trophectoderm formation.

Abstract

The trophectoderm (TE) is the first tissue to differentiate during the preimplantation development of placental mammals. It constitutes the outer epithelial layer of the blastocyst and is responsible for hatching, uterine attachment, and placentation. Thus, its formation is the key initial step that enables the viviparity of mammals. Here, we first describe the general features of TE formation at the morphological and molecular levels. Prospective TE cells form an epithelial layer enclosing an expanding fluid-filled cavity by establishing the apical-basal cell polarity, intercellular junctions, microlumen, and osmotic gradient. A unique set of genes is expressed in TE that encode the transcription factors essential for the development of trophoblasts of the placenta upon implantation. TE-specific gene expressions are driven by the inhibition of HIPPO signaling, which is dependent on the prior establishment of the apical-basal polarity. We then discuss the specific roles of RHO and ROCK as essential regulators of TE formation. RHO and ROCK modulate the actomyosin cytoskeleton, apical-basal polarity, intercellular junctions, and HIPPO signaling, thereby orchestrating the epithelialization and gene expressions in TE. Knowledge of the molecular mechanisms underlying TE formation is crucial for assisted reproductive technologies in human and farm animals, as it provides foundation to help improve procedures for embryo handling and selection to achieve better reproductive outcomes.

DOI:
https://doi.org/10.1530/REP-21-0478
Volume 164: Issue 4,
Pages:
R75–R86
Restricted access

Zinc as a non-hormonal contraceptive: an alternative to the copper intrauterine device (IUD)

K N Shankie-Williams, L A Lindsay, C R Murphy, and S N Dowland

In brief

The many side effects of current contraceptives leave a large proportion of women without adequate protection. This study shows that zinc, a highly physiologically compatible metal, provides effective long-acting reversible contraception in rats, without requiring the use of hormones.

Abstract

Long-acting and reversible contraceptives (LARC) are the most widely used form of female contraception worldwide; however, they have significant side effects that often result in early removal. Most LARCs are hormonal, but the use of exogenous hormones is not suitable for all women and causes side effects in many others. The copper IUD (CuIUD) is the only non-hormonal LARC, but a large proportion of users suffer severe side effects. This study proposes the use of zinc as a suitable alternative to the CuIUD. A rat intrauterine device (IUD) model was established to test the efficacy of a zinc IUD (ZnIUD) against a CuIUD. The IUD was surgically implanted into one uterine horn while the other remained untreated. Both the ZnIUD and CuIUD resulted in zero implantation sites which were significantly fewer compared to non-treated horns. Histological assessment revealed damage and inflammation in the endometrium of CuIUD-treated horns but only minor epithelial changes in ZnIUD-treated horns. This suggests ZnIUDs may not share the side effect profile of the CuIUD. To test the long-term efficacy of the ZnIUD, rats had a ZnIUD surgically implanted into both horns and cohoused with males for 3 months. These rats mated regularly but did not get pregnant, confirming long-term effectiveness. Reversibility of the ZnIUD was also established, as removal of the ZnIUD after 3 months resulted in no significant difference in the number of implantation sites between treated and untreated horns. This study demonstrated the contraceptive efficacy of zinc and its potential as a LARC.

DOI:
https://doi.org/10.1530/REP-22-0155
Volume 164: Issue 4,
Pages:
135–142 (8 total)
Restricted access

Cholesterol supports bovine granulosa cell inflammatory responses to lipopolysaccharide

Anthony D Horlock, Thomas J R Ormsby, Martin J D Clift, José E P Santos, John J Bromfield, and I Martin Sheldon

In brief

Bovine granulosa cells need to be cultured with serum to generate inflammation in response to bacterial lipopolysaccharide. This study shows that it is cholesterol that facilitates this lipopolysaccharide-stimulated cytokine secretion.

Abstract

During bacterial infections of the bovine uterus or mammary gland, ovarian granulosa cells mount inflammatory responses to lipopolysaccharide (LPS). In vitro, LPS stimulates granulosa cell secretion of the cytokines IL-1α and IL-1β and the chemokine IL-8. These LPS-stimulated inflammatory responses depend on culturing granulosa cells with serum, but the mechanism is unclear. Here, we tested the hypothesis that cholesterol supports inflammatory responses to LPS in bovine granulosa cells. We used granulosa cells isolated from 4 to 8 mm and >8.5 mm diameter ovarian follicles and manipulated the availability of cholesterol. We found that serum or follicular fluid containing cholesterol increased LPS-stimulated secretion of IL-1α and IL-1β from granulosa cells. Conversely, depleting cholesterol using methyl-β-cyclodextrin diminished LPS-stimulated secretion of IL-1α, IL-1β and IL-8 from granulosa cells cultured in serum. Follicular fluid contained more high-density lipoprotein cholesterol than low-density lipoprotein cholesterol, and granulosa cells expressed the receptor for high-density lipoprotein, scavenger receptor class B member 1 (SCARB1). Furthermore, culturing granulosa cells with high-density lipoprotein cholesterol, but not low-density lipoprotein or very low-density lipoprotein cholesterol, increased LPS-stimulated inflammation in granulosa cells. Cholesterol biosynthesis also played a role in granulosa cell inflammation because RNAi of mevalonate pathway enzymes inhibited LPS-stimulated inflammation. Finally, treatment with follicle-stimulating hormone, but not luteinising hormone, increased LPS-stimulated granulosa cell inflammation, and follicle-stimulating hormone increased SCARB1 protein. However, changes in inflammation were not associated with changes in oestradiol or progesterone secretion. Taken together, these findings imply that cholesterol supports inflammatory responses to LPS in granulosa cells.

DOI:
https://doi.org/10.1530/REP-22-0032
Volume 164: Issue 3,
Pages:
109–123 (15 total)
Restricted access

Effect of estradiol and IGF1 on glycogen synthesis in bovine uterine epithelial cells

Alexis Gonzalez, Malia D Berg, Bruce Southey, and Matthew Dean

In brief

Glucose is an important nutrient for the endometrium and embryo during pregnancy. This study shows that estradiol (E2)/IGF1 signaling stimulates glycogen synthesis in the uterine epithelium of cows, which could provide glucose when needed.

Abstract

Glycogen storage in the uterine epithelium peaks near estrus and is a potential source of glucose for the endometrium and embryos. However, the hormonal regulation of glycogen synthesis in the uterine epithelium is poorly understood. Our objective was to evaluate the effect of E2 and insulin-like growth factor 1 (IGF1) on glycogenesis in immortalized bovine uterine epithelial (BUTE) cells. Treatment of BUTE cells with E2 (0.1–10 nM) did not increase glycogen levels. However, treatment of BUTE cells with IGF1 (50 or 100 ng/mL) resulted in a >2-fold increase in glycogen. To determine if the uterine stroma produced IGF1 in response to E2, bovine uterine fibroblasts were treated with E2, which increased IGF1 levels. Immunohistochemistry showed higher levels of IGF1 in the stroma on day 1 than on day 11, which coincides with higher glycogen levels in the uterine epithelium. Western blots revealed that IGF1 treatment increased the levels of phospho-AKT, phospho-GSKβ, hexokinase 1, and glycogen synthase in BUTE cells. Metabolomic (GC-MS) analysis showed that IGF1 increased 3-phosphoglycerate and lactate, potentially indicative of increased flux through glycolysis. We also found higher levels of N-acetyl-glucosamine and protein glycosylation after IGF1 treatment, indicating increased hexosamine biosynthetic pathway activity. In conclusion, IGF1 is produced by uterine fibroblasts due to E2, and IGF1 increases glucose metabolism and glycogenesis in uterine epithelial cells. Glycogen stored in the uterine epithelium due to E2/IGF1 signaling at estrus could provide glucose to the endometrium or be secreted into the uterine lumen as a component of histotroph.

DOI:
https://doi.org/10.1530/REP-22-0040
Volume 164: Issue 3,
Pages:
97–108 (12 total)