• To purpose and outline
• To details of research

---

Purpose and Outline

---

Purpose of our research

　The purpose of our research is to elucidate the sexual and reproductive mechanims of mammals focusing on “hormone” as a keyword, and to provide feedback to the field. As a foundation of animal husbandry, we are working on the reproductive physiology of mammals with the goal of realizing efficient breeding of agricultural livestock and reducing the population of wild pests.

Declining fertility in cows

Crop damage caused by wild animals

---

Details of Research

---

1. Getting to the heart of the brain mechanisms that govern reproduction
2. Applying the brain mechanisms that govern reproduction
3. Search for male cattle-derived pheromones that stimulate reproductive functions in female cattle
4. Elucidation of a new mechanism that dominates the ovulation numbers in mammals
5. Mechanism of ovarian dysfunction caused by uterine inflammatory diseases in dairy cows
6. Identification of a disease-causing gene of the hereditary disease: incomplete fusion of the Müllerian ducts
7. Development of bovine embryos with high implantation potential

Getting to the heart of the brain mechanisms that govern reproduction

Hypothalamic-pituitary-gonadal axis and kisspeptin neurons

Follicular development and spermatogenesis in gonads (ovaries and testes) are promoted by gonadotropins (LH and FSH) secreted by the pituitary gland. LH and FSH are secreted into the blood in the pulsatile manner, which is induced by gonadotropin-releasing hormone (GnRH) pulses. The mechanisms by which GnRH pulses are secreted from GnRH neurons in the hypothalamus has long been unknown, but in recent years, it has been suggested that “KNDy neurons” in the hypothalamic arcuate nucleus are the center of GnRH pulse generation. KNDy neurons secrete three types of neuropeptides: kisspeptin, neurokinin B (NKB), and dynorphin A (Dyn). The functions of each neuropeptide have been elucidated as well: kisspeptin acts on GnRH neurons to induce GnRH secretion, NKB acts on KNDy neurons themselves and promote GnRH secretion, and Dyn suppresses GnRH pulses. Although GnRH and LH/FSH pulses are suppressed by the action of ovarian estrogen, KNDy neurons express estrogen receptors and have been suggested to be the direct targets of this negative feedback action of estrogen. KNDy neurons also exist in males and are thought to regulate the hypothalamic-pituitary-gonadal axis under the negative feedback action of testis-derived androgens. 
On the other hand, ovulation is a physiological mechanism unique to females. Oocytes in fully matured follicles are released outside the ovaries by ovulation and reach the uterus through the ovarian duct, which enables the females to get pregnant. It is the surge-like (large transient) secretion of LH that induces ovulation, and the LH surge is induced by the GnRH surge. Previous research suggests that the GnRH surge is controlled by kisspeptin neurons located in the hypothalamic regions anterior to KNDy neurons including preoptic area (POA) and anteroventral periventricular nucleus (AVPV). The GnRH/LH surge is induced by the action of high concentrations of estrogen derived from ovarian follicles which have matured into preovulatory stage. It is also suggested that POA/AVPV kisspeptin neurons, which express estrogen receptors, are the direct targets of this positive feedback effects of estrogen.

The mechanism of generation of GnRH pulse and surge

It is said that kisspeptin neurons directly receive estrogen secreted from ovary and transmit the signaling to GnRH neurons in a feedback way. On the one hand, when the low concentration of estrogen act on KNDy neurons, it would suppress the GnRH pulse and subsequently the LH/FSH pulse. On the other hand, when the high concentration of estrogen act on kisspeptin neurons located at the area of preoptic/anteroventral periventricular nucleus, it would activate the GnRH/LH surge. However, the mechanism that how estrogen regulates the activity of pulse and surge has not been elucidated clearly yet.We are now using rats and ruminants like cattle and goats as the model animals to analyze the mechanism that the generation of GnRH pulse and surge. In addition, considering that the establishment of cell lines is also essential for interpreting the mechanism that how reproduction function is regulated in ruminants at the cell level, we have successfully established a series of domestic animal-derived cell lines of arcuate nuclei neurons such as “goat KNDy neurons cell line”, “goat preoptic kisspeptin neurons cell line” and “goat GnRH neurons cell line”. And the establishment of above cell lines are all for the first time reported in the world. By using these cell lines, we are able to not only search for the factors regulating the kisspeptin neurons in ruminants but figure out the mechanism that how GnRH pulse and surge generation is regulated by kisspeptin neurons. 
*This research is sponsored by Grant-in-Aid for Scientific Research (B), 「KAKENHI」. “Identification of a novel ovary-derived feedback factor that regulates the activity of hypothalamus-pituitary” (2021-2024 year, principal investigator: Fuko Matsuda).

Interpretation of the energy-sensing mechanism that regulates the central reproduction system in the brain

Nutrition condition is deeply related to the maintenance of normal reproductive functions, including the development of sperm and follicles. For example, if the condition of fasting lasts for a long time, the secretion of gonadotropin, a substance that promotes the growth of sperm and follicles, would stop. Such suppression of reproductive function which is due to lack of energy is common in a wide variety of mammals. And it is also one of the major reasons causing the decreased fertility rate of dairy cows which is an urgent problem in the dairy industry. Therefore, in order to obtain knowledge related with the method of improving the fertility of dairy cows, we are trying to clarify the mechanism that how energy deficiency is sensed in the brain and subsequently reproductive function is suppressed. Currently, we are focused on the ependymal cells in the brain and implementing experiments that are based on the hypothesis that ependymal cells could sense the low energy condition and thus regulate the activity of neurons controlling the function of reproduction.

Applying the brain mechanisms that govern reproduction

Based on the basic knowledge obtained above, we aim to develop breeding control technology for domestic animals, wild animals, and zoo animals.

Clinical applications for reproductive performance

Reproductive efficiency is critical in the livestock industry. However, the reproductive performance of livestock, especially cattle, has been decreasing worldwide over the past few decades. Progress in our research would provide innovative perspectives on developing clinical applications for reproductive performance in livestock. Our team has been studying whether the promotion of NKB-NK3R signaling or attenuation of Dyn-KOR signaling could stimulate KNDy neurons and then recover suppressed follicular development.

Population management of wild and zoo animals

Search for male cattle-derived pheromones that stimulate reproductive functions in female cattle

In sheep and goats, exposure to male-derived pheromones has been proved to strongly induce ovarian functions, the so-called ‘male effect.’ Identification of male effect pheromones is expected to contribute to the improvement of reproductive efficiency of dairy cows. We aim to identify and elucidate the structure of the primer pheromones which induce female cattle ovarian function. This research is a collaboration project with the Laboratory of Veterinary Ethology (Prof. Yukari Takeuchi), The University of Tokyo.

Elucidation of a new mechanism that dominates the ovulation numbers in mammals

In mammals, a large number of follicles begin to develop during each estrous cycle, but only a handful of them reach ovulation and the others undergo atresia during development. The ovulation number (the number of follicles which ovulate in the end) is varied among animal species; 1 in cattle, horses, humans, and monkeys, 1-3 in goats and sheep, and about 10 in pigs. However, the mechanisms regulating the ovulation number remain largely unknown. We are searching for new factors that predominantly determine the ovulation number by analyzing bovine ovaries, and generating genetically modified mice and analyzing their phenotypes.

Mechanism of ovarian dysfunction caused by uterine inflammatory diseases in dairy cows

Uterine inflammatory diseases (endometritis) commonly occur in postpartum dairy cows, resulting in infertility. Infection of the uterus with gram-negative bacteria results in the detection of lipopolysaccharide (LPS) in the plasma and ovarian follicular fluid. To elucidate the mechanism of reproductive dysfunction caused by uterine inflammation, we are investigating the effects of LPS on ovarian function through clinical studies using postpartum cows and cell culture systems of oocytes or follicular cells.

Identification of a disease-causing gene of the hereditary disease: incomplete fusion of the Müllerian ducts

Our studies have elucidated that severe incomplete fusion of the Müllerian ducts significantly affected poor conception in Holstein cattle. Since genetic factors likely cause this disease, our team analyzes genomic data to identify disease-relevant regions. Identification of the responsible gene will provide a new standard for genetic evaluation schemes of dairy cattle. Selecting cattle with proper gene arrangement can potentially improve the reproductive efficiency of dairy herds.

Development of bovine embryos with high implantation potential

In vitro fertilization-embryo transfer (IVF-ET) is widely used to support the stable production of milk and beef. We are developing techniques to produce IVF embryos that can tolerate various stresses, such as postpartum negative energy balance and heat stress. Further, we are exploring for effective indicators to select embryos with high implantation potential using time-lapse cinematography.