Estrous Cycle and Its Hormonal Regulation

Contributor: Prof. Jogen C Kalita, Animal Physiology and Biochemistry Lab, Department of Zoology, Gauhati University

1. ESTROUS CYCLE AND ITS HORMONAL REGULATION PROF. JOGEN C. KALITA MSc ( Gau ), PhD (King’s College London) Professor in-Charge Animal Physiology & Biochemistry Lab. Dept. of Zoology, Gauhati University, Guwahati, Assam, India

2. After going through this PPT the students will be able to learn the following: Know about Estrous cycle and the cyclic nature of reproductive activities in female mammals Different phases of the estrous cycle in mammals They will know about physiological changes happening internally inside animals body can be assessed by simple external observation using vaginal smears technique (without using any invasive/aggressive methods) Know about the hormonal feedback that exists among hormones of Hypothalamus, Pituitary and Ovaries for regulation of the cycle Applied value of the earned knowledge in the field, animal husbandry and in case of farm animals (induced breeding etc.) LEARNING OUTCOMES

3. DEFINITION OF ESTORUS CYCLE & RELATED TERMINOLOGY A cycle is a repetition of the same event (s) at a definite interval of time. The Estrous or Oestrous Cycle is the reproductive cycle occurring in non-primate mammals. The repetition of morphological/physiological and behavioural changes occurs in female mammals at a definite interval of time. This duration of time interval differs in species. The Estrous cycle has four distinct phases, proestrus, estrus , metestrus and diestrus . During their reproductive life female mammals exhibit two types of reproductive cycles, Estrous cycle and Menstrual cycle. Human female and primates have Menstrual Cycle. Unlike estrous cycle the Menstrual Cycle has a distinct bleeding phase.

4. DEFINITION OF ESTORUS CYCLE & RELATED TERMINOLOGY We should not confuse with the term " estrus “. Estrus is just a phase of the Estrous Cycle. The word “estrus” means psychological heat period (psychic heat) of receptivity of female towards male for mating. Only during this estrus period of the cycle a female receives a male for mounting. Estrous cycle starts after sexual maturity in females and are interrupted by anestrous (absence of estrous ) phases or by pregnancies.

5. The word “ estrus ” was first used by Heape (1900). This is a Latin form of the Greek word “ oistros ” which means “ sexual season ”, “ gadfly ”, “ frenzy ”, “ sting ” or “ madness ”. Heape further named and defined the phases of the mammalian estrous cycle into proestrus, metestrus, diestrus and anestrus . Proestrus is the preparatory stage for an animal coming into heat. Estrus is the actual period of heat when females receive male for mounting. Metestrus which is a brief period characterized with decline of corpus luteum functions in the absence of conception when the activities of reproductive organs gradually subside, Diestrus which is a period of short rest during the breeding season, and Anestrus which a non-breeding period when reproductive organs are quiescent. HISTORICAL BACKGROUND

6. Proestrus : The first stage in the estrous cycle immediately before estrus characterized by development of both the endometrium and ovarian follicles. Estrus : The second stage in the estrous cycle immediately before metestrus characterized by a receptivity to a male and to mating, often referred to as "heat" or "in heat". Pheromones may also be secreted only at this stage of her cycle. Metestrus : The third stage in the estrous cycle immediately before diestrus characterized by sexual inactivity and the formation of the corpus luteum. Diestrus : The last stage in the estrous cycle immediately before the next cycle proestrus characterized by a functional corpus luteum and an increase in the blood concentration of progesterone. Anestrus : Not a stage in the estrous cycle, but a prolonged period of sexual rest where the reproductive system is quiescent (inactive). STAGES OF THE ESTROUS CYCLE The descriptions below refer to the "typical" estrous cycle Proestrus - Estrus - Metestrus - Diestrus

7. DIFFERENCES BETWEEN ESTROUS CYCLE AND MENSTRUAL CYCLE Estrous cycles are named for the cyclic appearance of behavioral sexual activity ( estrus ) that occurs in all mammals except for higher primates. Menstrual cycles , which occur only in human female and primates, are named for the regular appearance of menses due to the shedding of the endometrial lining of the uterus.

8. ESTROUS CYCLE MENSTRUAL CYCLE Estrous cycle occurs in Infra-primate female mammals only, for example rats, mice, cat, dog, cow, horse, pig etc. Menstrual cycle occurs In human female and primates Estrous cycle has its four distinct phases, proestrus , estrus, metestrus and diestrus , where receptivity to males is limited to estrus phase only. Menstrual cycle h as no such type of proestrus , estrus, metestrus or diestrus phases and female can mate with male during the entire cycle period. Estrous cycle is normally of short duration starting from 4-days Menstrual cycle duration is always of about a months (28±2 days) Estrous cycle has no distinct bleeding phase Menstrual cycle has a distinct bleeding phase towards the end of each cycle DIFFERENCES BETWEEN ESTROUS CYCLE AND MENSTRUAL CYCLE

9. ESTROUS CYCLE TYPES In different mammals during their reproductive life, Estrous cycles vary between species Polyestrous Animals: Estrous cycles occurring throughout the time of the year ( e.g , cattle, pigs, mice, rats). They can become pregnant without regard to the season of the year. Seasonally Polyestrous Animals: Animals that have multiple estrous cycles only during certain periods of the year ( e.g , horses, sheep, goats, deer, cats). They are classified as “ short day breeders ” when they present estrous cycles during autumn when day length is decreasing (goats and ewes) and “ long day breeders ” (mares and queens) when they show estrous mainly during spring, when day length increases. Monestrous Animals : Animals that have one estrous cycle per year ( e.g , dogs, wolves, foxes, and bear)

10. CHANGES IN REPRODUCTIVE PHYSIOLOGY DURING ESTROUS CYCLE Female mammals always exhibit different kind of behavioural, as well as morphological, histological, cytological, and physiological changes in the reproductive tract and also hormonal changes during each estrous cycle as stated below PROESTRUS Follicles in ovaries start developing further Estrogen , the main female sex hormones of ovary increases in circulation Estrogen start acting on its target, uterus and vascularity of the female reproductive tract increases Inside uterus endometrial thickness and glands of endometrium begin to grow Estrogen level becomes the highest (levels peak)

11. ESTRUS During this time of the cycle female allows male to mate (mounting) Estrogen in circulation slowly decreases LH from anterior Pituitary level increases to peak (surge occurs) Ovulation occurs after few hours of surge of LH or during the surge of LH Uterine motility becomes high with contractions moving toward the oviduct Sperm transport is optimal during this time if mating occurs and cervical mucus volume increases

12. METESTRUS Circulating Estrogen level becomes low Corpus hemorrhagicum present Ovulation may occur in some animals FSH increases, triggering growth of follicles Uterus contractions subside endometrial glands continue to grow and become coiled

13. DIESTRUS Progesterone secreted from ovaries become higher in circulation FSH level becomes low Uterus Secrets fluid but the volume of fluid decreases over time Contraction stop Corpus luteum regresses at the end of this period if female is not pregnant

14. STUDY OF ESTROUS CYCLE USING VAGINAL SMEARS IN RATS AND MICE BACKGROUND The estrous cycle in rats and mice averages 4–5 days and is a repetitive but dynamic process whereby different cell types appear and recede in waves throughout the cycle, reflecting changes in the levels of estradiol and progesterone secreted by the ovarian follicles and further changes occurring in the uterus, mainly endometrium, cervices, and vagina. STOCARD & PAPANICOLAOU (19I7) dealt with the maturation process in the Graafian follicles using the smear method with vaginal fluid in the guinea pig. ALLEN (1923) and HISAW (1947) put forwarded a theory on the oestrous cycle in the Muridae (largest family of rodents) . Estrous cycles are characterized by morphological changes in ovaries, the uterus and the vagina which occur during different phases in the cycle (Goldman et al ., 2007). Vaginal smear method remains as the gold standard till date to study estrous cycle in commonly used experimental animals.

15. VAGINAL SMEAR PROCEDURE Estrous cycle can be studied by collecting vaginal smears with a sterile cotton plugs putting in to the vagina or by a dropper. We in our Lab used a sterilized stainless steel wire loop having a diameter of about 3.5 mm was dipped in distilled water to create a diaphragm of water inside the loop. One drop of distilled water was added on the middle of a clean microscopic slide. The loop was then inserted into the vagina and rotated gently 2-3 times to collect the vaginal cells and finally placed onto the water drop and spread softly to make a smear. The slide was then allowed to air dry, fixed in methanol and stained for 10-15 min in Giemsa stain and observed under normal microscope. Four distinct cell associations of vaginal cytology appear at four different stages of the cycle.

16. Animal Proestrus Estrus Metestrus Diaestrus Mice(4-6 days) 24hrs 24hrs 12 60±4 Rat (4-5 days) 12hrs 12hrs 21hrs 57±1hrs Estrus cycle length in Rodents (Mice & Rat), Cora et. al., 2015

17. Fig: Appearance of cytological changes in four phases of estrous cycle in mouse The different phases of oestrous cycle are usually identified according to cell types observed in vaginal smears VAGINAL CYTOLOGICAL CHANGES DURING OESTROUS CYCLE & IDENTIFICATION OF THE PHASES

18. Phase of the cycle Cell types Proestrous Large rounded nucleated epithelial cells with distinct plasma membrane (PM) and clear cytoplasm. Estrus E ntirely of cornified cells, in high numbers and usually forming clumps and sheets. Cells are ells with granulated cytoplasm. Cells with no distinct boundaries (PM) and nucleus. Metestrous Large numbers of leucocytes and smaller numbers of mostly large, non-granular and non-nucleated epithelial cells. Diestrous Mainly of leucocytes but sometimes with quite variable numbers of epithelial and small cornified cells Table: Cytological changes during 4 different phases of estrous cycle

19. Estrous cycle is a hormonally controlled cycle of activity of the reproductive organs in female mammals. The follicular stage (growth of the Graafian follicles, thickening of the lining of the uterus, secretion of oestrogen, and ovulation, is succeeded by the luteal phase (formation of the corpus luteum and secretion of progesterone), followed by regression and a return to the first stage. The first half of the cycle is also termed as Follicular phase or Estrogen phase due to estrogen dominance and the second half of the cycle is called the Luteal phase, as corpus Luteum forms during this time or as Secretary phase or Progesterone phase. There are four major hormones involved in the oestrous cycle. These hormones include Follicle-stimulating hormone Luteinizing hormone Estrogen Progesterone HORMONAL REGULATION OF THE ESTROUS CYCLE

20. Fig: Relationship between circulating reproductive hormones, reproductive cycle stage, and vaginal cytological outcomes through different phases estrous cycle in female mouse. (Jessica G. et. al., 2018)

21. The cycle is regulated by the hypothalamic-pituitary-gonadal axis, which produces hormones that dictate reproductive events. The reproductive axis is composed of the hypothalamus, pituitary, and the ovary HORMONAL FEEDBACK DURING ESTROUS CYCLE Fig: Hypothalamic Pituitary gonadal Axis, the interaction of the structures and hormones involved within the oestrous cycle

22. The sequence of hormonal release essentially begins with the synthesis and release of GnRH from the hypothalamus. This polypeptide hormone is transported to the anterior pituitary through a highly specialized capillary network called the hypothalamo-hypophyseal portal system. GnRH functions to stimulate the anterior pituitary to produce and release FSH and LH. FSH and LH are transported through systemic blood circulation to the ovaries, where they initiate a series of morphological changes in the ovaries (ovarian folliculogenesis ) that lead to ovulation (and pregnancy if fertilization occurs). The primary hormones produced by the ovary are Estrogen and Progesterone. These hormones are transported by the blood stream to "target" tissues (mainly uterus and breast) to cause a development. As the follicle grows, more estrogen is produced. As increasing amounts of estrogen are released into the blood stream and travel to the anterior pituitary, it acts in a positive feedback fashion, stimulating pulsatile LH release. The uterus found in the pelvic cavity also contributes to reproductive control, as it produces prostaglandin F 2a (PGF 2a ).

23. Changes occurring in the mouse estrous cycle are evident in the animal’s physiology and anatomy. The cells lining the vagina of the female rat respond to the levels of circulating hormones and can provide a valuable marker of the stage of preparation of the ovary and uterus for mating and for ovulation. Vaginal smears taken on consecutive days over a period of time can provide detailed information on the oestrous cycle. The normal oestrous cycle in the rat usually follows a 4-day pattern and the varying characteristics of the cells in the smear allow the days of the cycle to be classified relative to the predicted time of ovulation. Ovulation occurs at approximately midnight after the pro-oestrous stage, when the females become receptive to the male. IMPORTANCE OF STUDY OF ESTROUS IN ANIMALS

24. The stages of the vaginal cell cycle will normally correlate with changes in the female reproductive organs e.g. ovary and uterus. This knowledge can be used in many lab experiments to study reproductive performances of animals under different situation and for testing drugs etc. and in the field of induced breeding technology. I n farm animals and animal husbandry the detection of estrus and ovulation time is very important for farmers to specifically apply mating at the specific time. IMPORTANCE OF STUDY OF ESTROUS IN ANIMALS

25. SUGGESTED READINGS Essential Reproduction (Essentials): Amazon.co.uk: Martin H. Johnson, 8 th Editon Knobi and Neil’s Physiology of Reproduction, Vol. 2, 4 th Edition The murine female intestinal microbiota does not shift throughout the estrous cycle Jessica G. et al., Published: July 16, 2018 https://doi.org/10.1371/journal.pone.0200729

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