Hanana IKEGAMI (JSPS Research Fellow, JSPS Research Fellow)

Chie Matani (Assistant Professor, Department of Biological Sciences)

Yoshitaka Oka (Professor Emeritus Professor, Department of Biological Sciences)

Key points of the presentation

• By analyzing neural activity using the whole brain of a genetically modified female medaka ^{(Note 1)}, we found that high frequency activity of GnRH1 neurons ^{(Note 2)} in the brain causes regular ovulation.
• We have shown that hormonal signals emitted by the mature ovary and some time signals signaling morning and evening are transmitted to the GnRH1 neuron, which activates this neuron.
• The mechanism in the brain that causes regular ovulation is thought to be common to many vertebrates other than mammals, and this discovery is expected to advance our understanding of the mechanisms in the brain that regulate ovulation in vertebrates.

Summary of Presentation

In vertebrate females, ovulation is generally repeated regularly when the ovaries reach maturity, and this timing is precisely regulated by various hormones in the body and the brain. As the ovaries mature, they secrete sex hormones. It is known that ovulation occurs when sex hormones act on the brain and the pituitary gland releases a large amount of gonadotropic hormone LH ^{(Note 3)} at one time (LH surge) ^{(Note 4)} as GnRH made in GnRH1 neurons in the brain is released in the pituitary gland. However, the mechanism in the brain that causes the massive release of GnRH and LH necessary for ovulation has remained largely unknown.

A research group at The University of Tokyo's Graduate School of Science used whole medaka brains labeled with genetically modified GnRH1 neurons to record the electrical activity of these neurons and analyzed the effects of sex hormones in detail. As a result, they discovered that both signals mediated by estrogen (E) ^{(Note 5)}, a sex hormone secreted by the ovary, and time signals are transmitted to GnRH1 neurons, resulting in a massive release of GnRH/LH, which is necessary for ovulation. This discovery is expected to advance our understanding of the mechanisms in the brain that regulate accurate ovulation in vertebrate females.

Announcement

Reproduction is one of the most important biological phenomena, and animals leave behind offspring. In most vertebrate females, once the ovaries have matured and the body is ready to reproduce, the ovaries repeatedly mature follicles and ovulate, and this cycle is called the reproductive cycle. The reproductive cycle is precisely controlled by various hormones in the body and the brain. It is generally known that the sex hormones released from the ovaries as the follicles mature act on the brain to produce GnRH neurons in the hypothalamus of the brain, which induce a massive release of GnRH into the pituitary gland at regular intervals, and that these signals cause a temporary massive release of LH from the pituitary gland, leading to ovulation (Figure 1). (Figure 1).

Figure 1: How ovulation is regulated in vertebrates
The release of GnRH from gonadotropin-releasing hormone (GnRH) neurons in the brain (GnRH surge) causes the release of luteinizing hormone (LH) from the pituitary gland (LH surge), which is carried in the blood stream to the ovaries and induces ovulation. In mature females, this sequence of events occurs regularly to form the reproductive cycle.

Until now, much research has been conducted on the mechanisms in the brain that control reproduction, particularly in mammals. Therefore, the mechanism by which GnRH neurons are activated to generate the LH surge necessary for ovulation in all vertebrates has remained a mystery.

The research group focused on the sex hormone E, which is secreted into the blood when the ovary matures, as the key to the mechanism by which the brain detects the development of follicles. This was inspired by the results of a previous study by this research group that showed diurnal variation in blood E levels in females (Ref. 1). Using medaka (Oryzias latipes), which has many advantages in reproductive neuroendocrinology experiments, such as easy genetic modification and regular spawning every morning, we conducted a detailed analysis of the effects of E by recording the electrical activity of neurons in whole medaka brains labeled with GnRH1 neurons through genetic modification. We also performed a histological analysis of where in the brain E acts.

First, using whole medaka brains, we recorded the neuronal activity (electrical signals) of GnRH1 neurons labeled with the fluorescent protein GFP ^{(Note 6)} by genetic modification, and found that the high-frequency neuronal activity of more than 6 times per second, which is required to induce the GnRH/LH surge (Ref. 2), occurs more in the evening than in the morning (Ref.2), which is required to induce GnRH and LH surges. In addition, neural activity of GnRH1 neurons in ovary-removed females was reduced in the evening, but neural activity was restored when ovary-removed females were fed an E-containing diet to increase blood E levels (Figure 2).

Figure 2: Effect of estrogen released from the ovary on the neural activity of GnRH neurons.
(A) In the morning, GnRH neurons showed low activity in ovariectomized and ovariectomized females, as well as in females fed estrogen-containing food, similar to the control group. On the other hand, in the evening, neural activity of GnRH neurons decreased with ovary removal and recovered with estrogen-containing feed. (B) In medaka with a one-day reproductive cycle (ovulation occurs daily), estrogen from the ovary activated GnRH neurons only in the evening. This may lead to the induction of regular ovulation.

In contrast, neither ovary removal nor feeding E-containing food altered the neural activity of GnRH1 neurons in the morning. These results indicate that the action of ovary-derived E in females occurs only in the evening. A similar analysis using males with low blood E levels throughout the day and fed an E-containing diet did not show the same fluctuations in neuronal activity observed in females, indicating that the above phenomenon is specific to females.

Furthermore, in order to clarify where in the brain E acts, we analyzed in which region of the brain the E-binding receptors are produced in neurons. The results suggest that among the three types of E receptors known in medaka, a type called Esr2a is produced and functions in GnRH1 neurons.

These results indicate that in female killifish, E signals released from the ovary during follicle maturation, together with time signals, are transmitted to GnRH1 neurons, which in turn enhance the neuronal activity of GnRH1 neurons. This mechanism is thought to allow the brain to sense that the follicle has matured. In other words, in order to excite GnRH1 neurons at the correct time, we found that in addition to E from the ovary, a time signal is also necessary. Since previous studies have suggested that multiple types of E receptors are required for ovulation to occur, it is possible that the pathway in the brain shown in (Fig. 3) is involved in the arousal of the neural activity of GnRH1 neurons. This discovery is expected to advance our understanding of how the brain regulates accurate ovulation in vertebrates. It is also expected that the results of this research will serve as a foothold for research into the development of new methods for increasing aquaculture.

Figure 3: Working hypothesis on how estrogen signaling and time signaling activate GnRH neurons.
Three types of receptors that bind estrogen are known in ectotherms. Of these, only estrogen receptor (Esr) 2a is found to be expressed on GnRH neurons themselves. It has also been reported that neighboring neurons with Esr1 extend their neurites directly to GnRH neurons (Ref. 3), and since medaka with loss of function of one of each of the three types of Esr can ovulate (Ref. 4), two or more of these types of Esr may be involved in the activation of GnRH neurons. The effect of estrogen is only observed in the evening. The fact that the effect of estrogen was observed only in the evening clearly indicates that time signals are also necessary to induce regular ovulation, but the identity of these signals and their mechanisms in the brain are the subject of future research.

References
1. Kayo D., Oka Y. and Kanda S. Gen Comp Endocrinol 285 (2020): 113272.
2. Hasebe M. and Oka Y. Endocrinol 158 (2017): 2603-2617.
3. Zempo B., Karigo T., Kanda S., Akazome Y. and Oka Y. Endocrinol 159 (2018): 1228-1241.
4. Kayo D., Zempo B., Tomihara S., Oka Y. and Kanda S. Sci Rep 9 (2019): 1-11.

Published Journals

Journal Name.	Journal of Neuroendocrinology
Title of Article.	Estrogen upregulates the firing activity of hypothalamic gonadotropin-releasing hormone (GnRH1) neurons in the evening in female medaka
Author(s)	Kana Ikegami, Sho Kajihara, Chie Umatani, Mikoto Nakajo, Shinji Kanda, Yoshitaka Oka* (University of Tokyo)
DOI Number	10.1111/jne.13101

Terminology

Note 1: Analysis of neural activity from the whole brain

A method of conducting rigorous neurophysiological experiments in which the whole brain is removed from a dish and kept in artificial cerebrospinal fluid to take advantage of the characteristics of the brain of small fish, while maintaining the same neural circuits as a live animal brain and the functional connections between the brain and pituitary gland. This method, which our laboratory developed ahead of others in the world, enables us to conduct experiments that are difficult to conduct in experimental animals such as mice. ↑up

Note 2 GnRH1 neurons

GnRH stands for Gonadotropin-Releasing Hormone, and GnRH1 neurons are neurons that produce and release GnRH1. In vertebrates, there are three genes, gnrh1-3, which arose mainly during evolution, but in medaka and other animals, it is known that GnRH1 encoded by gnrh1 is released in the pituitary gland, which in turn promotes the release of gonadotropin from the pituitary gland, resulting in ovulation. ↑up

Note 3 Gonadotropic hormone

A hormone secreted by the pituitary gland that causes gonadal development and ovulation through systemic blood circulation. There are two types: luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In vertebrates in general, ovulation is known to occur when LH is released in large amounts. ↑up

Note 4 GnRH/LH surge

The temporary release of a large amount of LH is called the LH surge, and it is known in vertebrates in general that the LH surge triggers ovulation in the ovary. On the other hand, GnRH surge is also thought to exist to trigger LH surge because GnRH released from the neurite terminals of GnRH neurons is released toward LH-producing cells in the pituitary gland, thereby promoting LH release. ↑up

Note 5 Estrogen (E )

A type of sex hormone generally referred to as female hormone. It is produced mainly from cholesterol in the ovaries of females. It is transported throughout the body via the blood circulation and causes female-specific secondary sexual characteristics via receptors to which estrogen binds (estrogen receptors). There are also neurons in the brain that have estrogen receptors, and estrogen is known to cause a specific response in those neurons. ↑up

Note 6 GFP

GFP is an acronym for green fluorescent protein, which is a fluorescent protein of the giant jellyfish, and became famous when Dr. Osamu Shimomura was awarded the Nobel Prize. By incorporating this gene downstream of the promoter of a specific gene in the host and introducing and expressing it in the host, only cells expressing the specific gene can produce GFP and be fluorescently labeled. ↑up