Research Topics of the Kowalewski Group
1. Hypoxia & Steroidogenesis
How does oxygen deprivation influence hormone production in reproductive organs?
- The hypoxia-inducible transcription factor HIF1α regulates steroid biosynthesis by controlling the STAR protein.
- Oxygen deprivation alters mitochondrial dynamics and influences hormone production.
- Experimental models: murine and bovine steroidogenic cells in vitro.
Summary:
Our research investigates how physiologically reduced oxygen concentrations ("physioxia") and hypoxia-dependent signalling pathways regulate steroid hormone production in steroidogenic cells of the gonads. Particular emphasis is placed on the hypoxia-inducible transcription factor HIF1α, a key cellular oxygen sensor.
Using granulosa cell models, we demonstrated that HIF1α positively regulates the expression of the Steroidogenic Acute Regulatory protein (STAR) and progesterone synthesis. This effect is mediated through cAMP/PKA-dependent transcription factors, including cJUN, CREB and the co-activator CBP. Furthermore, we identified HIF-stabilising enzymes, particularly prolyl hydroxylases (PHDs), as important regulators of HIF1α activity. Our findings demonstrate that precisely balanced HIF1α activity is essential and results in a biphasic regulation of STAR expression: both insufficient and excessive HIF1α activity can impair steroidogenesis.
To improve molecular biological analyses, we also developed validated reference gene sets for hypoxia-related studies in steroidogenic tissues and cell lines. In addition, we characterised the spatial and temporal expression patterns of HIF1α and its regulators in the bovine corpus luteum during the luteal phase and under inflammatory conditions following LPS stimulation. These studies revealed important links between oxygen sensing, vascular inflammation and progesterone-dependent fertility processes.
- Eilers Smith O et al. (2025) – Optimizing gene expression analysis under hypoxia: identification and validation of novel reference genes in steroidogenic tissues and cell lines
- Gomes LAB et al. (2025) – Dynamic Regulation of HIF1α and Oxygen-Sensing Factors in Cyclic Bovine Corpus Luteum and During LPS Challenge
- Lanfranchi B et al. (2022) – Transcriptional regulation of HIF1α-mediated STAR expression in murine KK1 granulosa cell line involves cJUN, CREB and CBP-dependent pathways
- Gysin T & Kowalewski MP (2021) – The involvement of hypoxia-inducible factor 1α (HIF1α)-stabilising factors in steroidogenic acute regulatory (STAR) protein-dependent steroidogenesis in murine KK1 granulosa cells in vitro
- Kowalewski MP et al. (2015) – The role of hypoxia and HIF1α in the regulation of STAR-mediated steroidogenesis in granulosa cells
• To produce sex hormones such as progesterone, the body requires an important regulatory protein called STAR.
• STAR transports cholesterol to the site where steroid hormones are synthesised. Without STAR, steroid hormone production is severely impaired.
The role of HIF1α:
HIF1α acts as a cellular oxygen sensor. It responds to oxygen availability and thereby regulates hormone production.
Three possible scenarios:
1. Too little HIF1α: STAR expression is reduced, leading to decreased hormone production.
2. Optimal HIF1α activity: STAR functions efficiently, allowing normal progesterone production.
3. Too much HIF1α: STAR is suppressed again, resulting in reduced hormone production.
In addition, HIF1α interacts with co-regulatory proteins such as cJUN, CREB and CBP.
2. The Role of HIF1α and Hypoxia During IVP
How do oxygen availability and HIF1α influence oocyte quality and embryonic development in vitro?
- HIF1α regulates key processes during the maturation of cumulus–oocyte complexes (COCs).
- Physiological hypoxia promotes communication between cumulus cells and the oocyte.
- Both inhibition and excessive activation of HIF1α can impair embryonic development.
- Experimental models: bovine cumulus–oocyte complexes (COCs), in vitro maturation (IVM) and in vitro fertilisation (IVF).
Summary:
By investigating the role of HIF1α during the in vitro maturation of bovine cumulus–oocyte complexes (COCs), we demonstrated that pharmacological inhibition of HIF1α impairs COC function. Specifically, inhibition of HIF1α activity and/or expression resulted in reduced cumulus expansion, decreased steroidogenic activity and impaired nuclear maturation of oocytes. These effects led to significantly lower blastocyst rates following in vitro fertilisation (IVF), demonstrating that HIF1α signalling is essential for normal communication between cumulus cells and the oocyte as well as for the acquisition of oocyte developmental competence.
Conversely, using a targeted pharmacological approach, we demonstrated that controlled, moderate stabilisation of HIF1α during in vitro maturation—mimicking the physiological hypoxic environment of the ovarian follicle—improves and stabilises blastocyst yield. In contrast, excessive stabilisation of HIF1α proved detrimental. Our findings therefore demonstrate the existence of a narrow biological window in which moderate activation of HIF1α enhances oocyte developmental competence, whereas both insufficient and excessive HIF1α activity negatively affect embryonic development.
Overall, these findings highlight the importance of physiological follicular hypoxia as a key determinant of oocyte quality and suggest that precise modulation of HIF1α represents a promising strategy for the biological optimisation and improvement of bovine IVF procedures.
- Gübeli M et al. (2026) – Targeted hypoxia-inducible factor 1-alpha (HIF1A) stabilization during in vitro maturation of bovine cumulus-oocyte complexes increases blastocyst rates
- Turhan A et al. (2021) – Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro
• Oocytes naturally mature within the ovary under conditions of relatively low oxygen availability.
• To respond to these conditions, cells rely on a biological oxygen sensor called HIF1α.
• HIF1α enables cumulus cells to provide optimal support for the oocyte throughout its maturation.
Three possible scenarios:
1. Too little HIF1α:
The cells are unable to adequately adapt to the low-oxygen environment. Communication between cumulus cells and the oocyte is disrupted, resulting in reduced oocyte quality and developmental competence.
2. Optimal HIF1α activity:
The oocyte matures under physiological conditions. Communication between cumulus cells and the oocyte functions optimally, providing the basis for successful fertilisation and embryonic development.
3. Too much HIF1α:
Excessive activation of HIF1α disrupts the natural balance of cellular signalling pathways. As a consequence, essential developmental processes may be impaired, leading to compromised embryonic development.
3. Embryo–Maternal Communication
How do the embryo and the uterus communicate in the bitch?
- An intensive molecular dialogue between the embryo and the uterus is established before and during implantation.
- Growth factors, hormones and immune mediators regulate communication between the embryo and the endometrium.
- The extracellular matrix (ECM) undergoes targeted remodelling to facilitate embryo implantation and placental development.
- Immunological mechanisms ensure the delicate balance between maternal tolerance of the embryo and the maintenance of local immune defence.
- Novel in vitro models enable these processes to be investigated under controlled experimental conditions.
Summary:
Embryo–maternal communication during implantation and early pregnancy in the bitch is orchestrated by a complex interplay of growth factors, hormones, prostaglandins, immune mediators and components of the extracellular matrix (ECM). These signals regulate the interaction between the early embryo and the maternal endometrium, thereby establishing the conditions required for successful pregnancy establishment.
A major focus of our research is the development and application of innovative in vitro models that recapitulate key events of early pregnancy. These include primary endometrial stromal cells. These models enable the investigation of molecular signalling pathways regulating implantation, tissue remodelling, immune modulation and embryo–maternal communication.
Another major focus is the characterisation of the distinct effects of seminal and embryo-derived signals on the maternal uterus. We investigate how hormonal and immunological pathways are activated prior to implantation and how the embryo actively modulates the uterine environment to establish the conditions required for successful pregnancy.
By combining cell culture models, molecular biology approaches and state-of-the-art omics technologies, we gain new insights into the biological mechanisms underlying implantation, embryo–maternal communication and the early immunological adaptation of the uterus. The overall aim of these studies is to improve our understanding of the fundamental mechanisms governing successful pregnancy establishment and to generate new insights into reproductive biology.
- Adwell Mushati K et al. (2026) – Modulation of uterine responses in dogs: The divergent effects of semen and embryonal signals on steroidogenic receptors and selected immune system-related factors
- Tavares Pereira M et al. (2025) – Placental interleukin 1β signaling in canine pregnancy: differential effects during and after decidualization in vitro
- Kowalewski MP (2023) – Advances in understanding canine pregnancy: Endocrine and morpho-functional regulation
- Tavares Pereira M et al. (2022) – Transcriptomic profiling of canine decidualization and effects of antigestagens on decidualized dog uterine stromal cells
- Kazemian A et al. (2022) – Antigestagens Mediate the Expression of Decidualization Markers, Extracellular Matrix Factors and Connexin 43 in Decidualized Dog Uterine Stromal (DUS) Cells
- Tavares Pereira M et al. (2021) – Selected Uterine Immune Events Associated With the Establishment of Pregnancy in the Dog
- Kowalewski MP et al. (2020) – Canine conceptus-maternal communication during maintenance and termination of pregnancy, including the role of species-specific decidualization
- Graubner FR et al. (2020) – Canine decidualization in vitro: extracellular matrix modification, progesterone mediated effects and selective blocking of prostaglandin E2 receptors
- Nowak M et al. (2019) – Gene expression profiling of the canine placenta during normal and antigestagen-induced luteolysis
- Nowak M et al. (2018) – Luteal and hypophyseal expression of the canine relaxin (RLN) system during pregnancy: Implications for luteotropic function
- Nowak M et al. (2017) – Functional implications of the utero-placental relaxin (RLN) system in the dog throughout pregnancy and at term
- Graubner FR et al. (2017) – Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species
- Kautz E et al. (2015) – In vitro decidualisation of canine uterine stromal cells
- Kautz E et al. (2014) – Expression of genes involved in the embryo-maternal interaction in the early-pregnant canine uterus
• An intensive molecular dialogue between the embryo and the uterine lining begins only a few days after fertilisation.
• The embryo releases signalling molecules that prepare the uterus for implantation and create the conditions required for successful pregnancy.
• At the same time, the maternal immune system adapts. It must recognise and tolerate the embryo as a new organism while maintaining its essential protective function against pathogens.
• Our research investigates how hormonal, immunological and molecular signalling pathways coordinate this dialogue between the embryo and the mother, thereby enabling successful pregnancy establishment.
Our research focuses on:
– How the embryo prepares the uterus for implantation
– Which immune mediators and signalling molecules regulate early pregnancy
– How the uterus adapts to the presence of the embryo
4. Decidualization & New Concepts of Canine Placentation
What role do decidual cells play in the placenta of the bitch?
- The bitch exhibits pronounced decidualization of the uterine lining.
- Decidual cells are hormone-dependent maternal cells with a central role in maintaining pregnancy.
- Progesterone receptors in the canine placenta are predominantly expressed by decidual cells.
- Extracellular vesicles (EVs) mediate important signals between maternal decidual cells and fetal trophoblast at the fetomaternal interface.
- Modern 3D reconstructions reveal complex spatial interactions between maternal decidual cells and fetal trophoblast.
- Current investigations using single-nucleus RNA sequencing (snRNA-seq) enable high-resolution characterisation of individual cell populations and their molecular functions at the fetomaternal interface.
- These findings support an expanded understanding of the canine placental barrier and highlight the particular importance of maternal decidual cells as active components of the fetomaternal interface. They further point to the relevance of a deciduo-chorial interface as a functionally important component of canine placentation.
Summary:
Our research investigates the origin, differentiation and function of decidual cells in the canine placenta. Using molecular biological, ultrastructural and three-dimensional analyses, we demonstrated that decidual cells represent a highly specialised maternal cell population with a central role in progesterone action, maintenance of placental function and tissue remodelling processes around parturition.
A particular focus is placed on the role of extracellular vesicles (EVs) as a communication mechanism between maternal decidual cells and fetal trophoblast. These nanoscale signal carriers transport proteins, lipids and regulatory RNA molecules and can thereby influence cell function, differentiation and communication at the fetomaternal interface.
Current investigations using serial scanning electron microscopy (SEM) and three-dimensional reconstructions have revealed extensive physical interactions between maternal decidual cells and fetal trophoblast cells at the fetomaternal interface. These findings support an expanded understanding of the canine placental barrier and point to the particular importance of a deciduo-chorial interface as a functionally relevant component of canine placentation.
Building on these findings, we are currently using state-of-the-art single-nucleus RNA sequencing (snRNA-seq) to investigate the cellular composition and functional specialisation of the canine placenta at single-cell resolution. The aim is to identify novel cell populations as well as their molecular signalling pathways and interactions at the fetomaternal interface, thereby improving our understanding of the mechanisms underlying placentation and pregnancy maintenance.
- Kowalewski MP (2026) – Morpho-functional uterine and placental dynamics during establishment and maintenance of canine pregnancy, from early trophoblast invasion to parturition
- Corte GM et al. (2025) – Morphological and functional aspects of the canine placental barrier: evidence supporting a deziduo-chorial model
- Kowalewski MP (2023) – Advances in understanding canine pregnancy: Endocrine and morpho-functional regulation
How does the connection between mother and puppy work?
• During pregnancy, specialised maternal cells, known as decidual cells, develop in the uterus. They support placental development and help maintain pregnancy.
• These cells are in close contact with fetal trophoblast cells and together form the interface between mother and offspring.
• Part of this communication is mediated by tiny particles called extracellular vesicles (EVs). They transport proteins, RNA and other signalling molecules between cells and coordinate important processes during placental development.
• Modern 3D reconstructions show that this contact zone is far more complex than previously assumed and consists of a closely interconnected network of maternal and fetal cells.
• Using state-of-the-art single-nucleus RNA sequencing (snRNA-seq), we can now even investigate the activity of individual cell nuclei. This allows us to identify which cell types are present in the placenta, which genes they use and how they communicate with each other.
• These findings help us to better understand the development and function of the placenta as well as the mechanisms underlying a healthy pregnancy.
5. Reproductive Biotechnologies in Horses and Cattle
How do hormonal, paracrine and inflammatory signals in the ovary and uterus influence oocyte quality, embryo production and fertility in cows and mares?
- A healthy and finely balanced intrafollicular environment is essential for the acquisition of oocyte developmental competence.
- Oocyte-secreted factors regulate follicular growth and maturation and are considered key regulators as well as potential therapeutic targets.
- Our research focuses on oocyte-secreted factors (GDF9, BMP15), glucocorticoids, steroid hormones and anti-Müllerian hormone (AMH) as biomarkers of ovarian reserve and IVF/ICSI success.
- Linking cellular mechanisms within the follicle to clinically relevant parameters provides new opportunities to optimise fertility and embryo production in veterinary practice.
Summary:
Our research focuses on how the microenvironment of the ovary and uterus determines oocyte developmental competence and ultimately the fertility of cows and mares. It demonstrates that the oocyte does not mature in isolation but is embedded within a highly coordinated network of granulosa cells, cumulus cells, follicular fluid, endocrine signals and inflammatory mediators that profoundly influence its quality.
A central role is played by oocyte-secreted factors of the TGF-β superfamily, such as GDF9 and BMP15, which regulate steroidogenesis, granulosa cell proliferation and apoptosis through paracrine signalling pathways, thereby controlling the transition from the growing follicle to the preovulatory follicle. At the same time, our data demonstrate that glucocorticoids are locally fine-tuned within the follicle and influence gene expression in cumulus–oocyte complexes without altering maturation rates, indicating that these "stress hormones" form an integral part of the physiological maturation programme.
Within this highly sensitive environment, metabolic and inflammatory stress often act silently but with profound consequences. Subclinical ketosis, mastitis and endometritis impair the morphological quality of oocytes and embryos and increase the incidence of abnormal cleavage patterns, even when conventional parameters such as cleavage and blastocyst rates initially remain unaffected. In addition, inflammatory mediators such as lipopolysaccharide compromise developmental competence but can be functionally counteracted by targeted supplementation with GDF9 during in vitro maturation (IVM), opening new therapeutic avenues for stabilising the ovarian microenvironment.
At the systemic level, these studies link intrafollicular processes to endocrine biomarkers such as AMH, oestradiol and progesterone, which provide valuable indicators of ovarian reserve and expected embryo yield. AMH has emerged as a robust biomarker of the functional follicular pool and is associated—depending on species and parity—with insemination intervals, embryo numbers and superovulatory response, while oestradiol and progesterone profiles help identify the optimal timing for OPU-IVP and OPU-ICSI procedures.
Taken together, these studies depict a highly dynamic reproductive axis in which oocyte-driven signalling pathways, endocrine profiles and animal health interact to determine the progression from follicular maturation to successful pregnancy in cows and mares.
- Vernunft and Scarlet (2026) – Application of Anti-Müllerian Hormone in Regulation of Ovarian Function and Its Practical Relevance for Fertility and Embryo Production in Cows and Mares
- Scarlet D et al. (2026) – Growth Differentiation Factor 9 Supplementation Restores the Lipopolysaccharide‐Induced Negative Effects on Bovine Oocyte Developmental Competence
- Samie KA et al. (2025) – Roles of GDF9 and BMP15 in equine follicular development: in vivo content and in vitro effects of IGF1 and cortisol on granulosa cells
- Scarlet D et al. (2025) – Endocrine profile and OPU-ICSI outcomes in mares: a comparative study
- Scarlet D et al. (2024) – Exogenous FSH/LH modulates TGF beta signaling genes in granulosa cells of Simmental heifers without affecting IVP results
- Serbetci et al. (2024) – Impact of negative energy balance and postpartum diseases during the transition period on oocyte quality and embryonic development in dairy cows
- Samie KA et al. (2023) – Effects of cortisol on granulosa cell function during follicle maturation in the horse
- Scarlet D et al. (2023) – Relationship between Clinical Uterine Findings, Therapy, and Fertility in the Mare
- Scarlet D et al. (2016) – Glucocorticoid metabolism in equine follicles and oocytes
• Growth factors such as GDF9 and BMP15, together with hormones and inflammatory mediators, determine how well the oocyte develops and whether it can ultimately give rise to a healthy embryo.
• Diseases such as inflammation or metabolic disorders can disturb this highly sensitive environment and thereby impair fertility.
• Using modern reproductive biotechnologies, we investigate how these processes can be improved—for example by optimising in vitro maturation (IVM), in vitro embryo production (IVP), as well as OPU-IVP and OPU-ICSI procedures in cows and mares.
• Our ultimate goal is to improve oocyte and embryo quality, enhance fertility and provide new scientific foundations for modern reproductive medicine and animal breeding.