Team:IISER-Tirupati India/Background

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INDEX

Beginning of OviCloak

• The Reproductive System
• Menstrual Cycle
• Fertilisation
• Ovastacin
• References

Conventional scientific literature only uses the terms “female” and “male” with respect to gonads, gametes and even the reproductive system as a whole. This is an attempt of our team to make scientific literature more inclusive of gender diverse and intersex individuals. While citing, the conventional term female has been replaced with “uterus owner”/ “menstruators/people with uteruses'' according to the context and the term “male” has been replaced with “people who ejaculate semen''.

Beginning of OviCloak

This page lays the foundational stone on which OviCloak has been designed. The idea behind OviCloak starts from basic high school concepts of the reproductive systems supplemented with more details to incorporate synthetic biology to find the solution.
We begin with revisiting the reproductive system of menstruators to understand the environment where our contraceptive is designed to work. Then we gain insight into the menstrual cycle to understand the regulatory aspects in our idea. Followed by the most important details of fertilisation which has been augmented with our literature survey and gives the significance and reasoning of how we chose our protein of interest and target to design a contraceptive. To answer the question of ‘how we incorporate synthetic biology in our project’, we acknowledge the presence of our friends down there and take their help to actualise our project.

The Reproductive System of Uterus Owners

The reproductive system of uterus owners consists of both internal and external sex organs helping in reproduction. It is immature at birth and develops at puberty to produce gametes and hormones and carry a fetus to full term. The internal sex organs include the ovaries, fallopian tube, uterus, and vagina. The external sex organs (genitals) are the organs of the vulva which include the labia, clitoris, and vaginal opening. The details of the internal sex organs are illustrated below.

• REPRODUCTIVE SYSTEM
• OVARY
• FALLOPIAN TUBE
• UTERUS
• CERVIX
• VAGINA

Fig 1. The reproductive system

Fig. 1.1 Diagrammatic representation of ovary

The ovary is the gonad found in the reproductive system of uterus owners, located beside the lateral wall of the uterus, one on each side. They produce ovum and hormones responsible for secondary sexual characteristics. During ovulation of the menstrual cycle, typically, one of the two ovaries releases an egg in each menstrual cycle. The developing oocytes (ovum) mature in the fluid-filled follicles. Upon ovulation, the oocyte from the mature follicle, the Graffian follicle, will be released into the fallopian tube, and the follicular cells give rise to a structure called corpus luteum [1].

Fig.1.2 Diagrammatic representation of the fallopian tube

Fallopian tubes or oviducts are tube-like structures that stretch from the ovaries to the uterus. The distal end of the fallopian tube opens to the ovary and the proximal opening to the uterus, at the uterotubal junction. There are three other regions of the fallopian tube: isthmus, ampulla, and infundibulum. During ovulation, the ovum released will enter the infundibulum of the fallopian tube for fertilization. After fertilization, the zygote reaches the uterus for implantation, which is facilitated by a specialized ciliated columnar epithelium of the fallopian tube [2].

The details of various regions of the fallopian tubes are given below:

FIMBRIA:

Fimbriae are finger-like projections on the distal end of the fallopian tube. During ovulation, these structures are responsible for safely capturing the oocyte and releasing it into the fallopian tube [2].

INFUNDIBULUM:

The infundibulum is the most proximal segment of the fallopian towards the ovary. It is a funnel-shaped structure and possesses fimbriae. During ovulation, fimbriae come close to the ovary to capture the oocyte and release it into the infundibular region of the fallopian tube [3].

AMPULLA:

The ampulla of the fallopian tube is the region between the infundibulum and isthmus. After ovulation, travels to the ampullary region via infundibulum. The significance of this region is, in humans, this part is generally the site of fertilization. This leads to the immediate completion of meiosis II of the oocyte[2].

ISTHMUS:

The isthmus is a short caudal segment of the fallopian tube that connects the utero-tubal junction. After fertilization, the zygote enters this region, and the development of the preimplantation embryo takes place [2].

Fig 1.3 Diagrammatic representation of the uterus

Uterus is a secondary sex organ in mammals and functions as a major hormone-responsive organ [4].

Anatomically, the uterus is divided into four regions: fundus, corpus, cervix, and cervical canal. The fundus - uppermost rounded portion, the corpus is the body. The lower part of the uterus is the cervix leading to the cervical canal, which opens to the vagina.

After fertilization, the embryo implants to the uterus where it develops into a fetus during the gestation period [5].

Fig 1.4 Diagrammatic representation of the cervix

The cervix is the lower, narrower part of the uterus which bulges through the anterior wall of the vagina. After sexual intercourse, sperm enter the uterus via the cervical canal [5].

Fig 1.5 Diagrammatic representation of the vagina

The vagina is a muscular region of the genital tract of uterus owners which is elastic in nature[6]. In humans, it extends from the outer vaginal opening vulva to the cervix. The vagina channels for menstrual flow, sexual intercourse, and childbirth.

MENSTRUAL CYCLE 

The menstrual cycle coordinates the production and release of the ovum and sex hormones to govern fertilization and embryo implantation by preparing and maintaining the uterus lining. Menarche, the onset of the first period, usually happens around twelve years and menopause, the end of the period, happens around fifty years of age. Between these years, the cycle (in an ideal case) is concurrent and coordinated and usually repeated every 28-30 days in response to hormone levels [6].

There are four stages of the menstrual cycle - Menstruation, Follicular, Ovulation, and Luteal phases, briefly described along with the hormonal variations [6].

Fig 2. The figure shows the menstrual cycle in an average uterus owner. The X-axis represents the number of days in the menstrual cycle according to different phase divisions and the Y-axis represents the different components of the menstrual cycle (from bottom to top) a. Uterine cycle showing the variation of thickness of the endometrium. b. Ovarian hormones, estrogen, and progesterone variation. c. Anterior pituitary hormones LH and FSH levels. d. Ovarian cycle showing the structural variation in Graafian follicle

Menstruation

When the corpus luteum degenerates, the estrogen and progesterone levels go down, the endometrium layer, which was growing thicker throughout the menstrual cycle starts to shed which is called menses. Menstruation happens from day 0 to day 5 of the menstrual cycle. The usual duration of the menstrual flow is 3-5 days in an average menstruator. Menstrual blood contains tissue debris, prostaglandins, and relatively high amounts of fibrinolysis from endometrial tissue [6].

Follicular phase or Proliferative phase

The follicular phase occurs from day 0 to day 14 of the menstrual cycle, for an average cycle length of 28 days. [6] During this phase, the follicles in the ovary mature from the primary follicle to a fully mature Graafian follicle. In this phase, Follicle-Stimulating Hormone (FSH) level rises, stimulating a few ovarian follicles and on the basis of the number of FSH receptors on the cells, Luteinizing Hormone (LH) stimulates the subsequent development of the dominant ovarian follicle. The follicle that reaches maturity is called the Graafian follicle, and it contains the ovum [7].

Ovulation 

Ovulation is the rupture and releases the dominant follicle from the ovary into the fallopian tube. For an average menstrual cycle of 28 days, ovulation occurs on day 14 [6].

During the follicular phase, granulosa cells produce estrogen and this continuous elevation in estrogen levels for two days results in the LH surge [8,9]. This LH surge leads to an increase in the production of progesterone in the tubal fluid where the initial concentration is approximately 1 nM in the follicular phase.

Luteal phase or Secretory Phase

The luteal or secretory phase occurs around day 14 to day 28 of the menstrual cycle after ovulation. During this phase, Graafian follicles cells give rise to the corpus luteum that increases the production of estrogen and progesterone [6]. The progesterone level rises as high as approximately 40 nM in tubal fluid during day 20, causing negative feedback to the anterior pituitary to decrease FSH and LH, reducing estrogen and progesterone levels to less than half the amount towards the end of the luteal phase [6,9].

This change in concentration of progesterone can be detected using a specific transcription factor. This helps to regulate the production of our protein of interest with respect to the regular menstrual cycle of the user. 

The ovum that is released from the ovary, enters the fallopian tube ready for fertilisation.

FERTILIZATION

The gametogenesis process leads to the production of sperm in people who ejaculate semen and ovum in people with ovaries.

Fig 3. Diagrammatic representation of Ovum

The ovum is surrounded by a spherical collection of cells known as ovarian follicles consisting of cumulus oophorus, granulosa, corona radiata and zona pellucida [10].

The fusion of sperm and ovum leads to fertilization. 

Fig 4. Diagrammatic representation of fertilisation

During coitus or artificial insemination, the sperms are ejaculated at the vagina, they swim through the uterus and reach the fallopian tube, near the ampulla.

The oocytes have membrane-bound organelles in the oolemma called cortical granules. Upon fertilization, cortical granules undergo exocytosis to release about 4000 cortical granules (CG) [11,12] during the cortical reaction [13] including Sperm Acrosomal SLLP1 Binding (SAS1B) protein or ovastacin. This protease ovastacin causes physico-chemical changes in the extracellular matrix surrounding the ovum [14], called the Zona Pellucida (ZP). It cleaves Zona Pellucida 2 (ZP2) glycoprotein component of ZP (120 kDa) into two fragments (90 kDa + 30 kDa) [15] at a conserved cleavage site (167LA ↓DE170) [16] which gives rise to hardening of the ZP layer. Zona Pellucida Hardening (ZPH) renders the outer layer of the ovum impermeable for further sperms, thus preventing polyspermy [17]. This concept of protease and substrate protein-specific cleavage gave us the correct combination of a contraceptive molecule or our protein of interest, ovastacin and the molecule 's target, the substrate ZP2.

Fig 5. Interaction between ovastacin and ZP2

While designing a contraceptive, specificity and effectiveness were the key factors to be considered first. Ovastacin ticked off both criteria. The ovum uses this protein to cloak itself from several invading sperms, and we chose to work with this very mechanism of ovum with a twist! We preponed the process of cloaking the ovum using ovastacin even before the entry of the first sperm, with the help of our genetically modified commensal bacteria.

Ovastacin:

The expression of ovastacin protein is localized on the plasma membrane of the oocyte, it has a specific target on the extracellular matrix of the ovum for its proteinase activity and its non-toxic nature makes this protein a potential contraceptive molecule.

The SAS1B protein or ovastacin is a member of the astacin family of metalloproteinases. It is a binding partner in the oolemma of oocytes for the intra-acrosomal sperm protein called SLLP1 (Sperm Lysozyme-Like Protein) [18]. In the reproductive system, SAS1B translation is restricted to the tissues at the ovary and oocytes of the reproductive system. It first appears in ovarian follicles during the transition of primary-secondary follicles in the form of zymogen in humans and is released from cortical granules during the cortical reaction in an activated form after getting cleaved [18]. A part of the C-terminal domain of ovastacin remains attached to the oolemma, while the N-terminal active ovastacin domain is secreted [19]. The secreted active protease acts specifically on ZP2 as it is a target for cleavage [20]. Thus this protease can act as an ideal candidate to be used for a novel reversible contraceptive strategy for uterus owners.

In order to explore the potential use of ovastacin as a contraceptive molecule, we turned towards synthetic biology when we found evidence of the presence of commensals in the reproductive tract through scientific literature.

Commensals in the reproductive system of uterus owners:

Fig 6. Commensal colonization in fallopian tube

Humans are not alone, commensal bacteria colonize many organ systems in the human body, and the reproductive organs are no exception. While the vaginal microbiota has been well established, there has been a general assumption that the upper genital tract is a sterile environment [21]. But, recently using new genomic technologies, scientists have identified specific patterns of microbiota colonizing the fallopian tubes and ovaries. The taxa found in fallopian tubes include Lactobacillus spp, Staphylococcus spp, Enterococcus spp, Burkholderia spp and Propionibacterium spp [21,22]. The presence of these commensal bacteria gives an untapped potential to use this for the benefit of human beings. However, more functional characterization of the microbiota of the upper reproductive tract shall help solve several problems with the reproductive health of uterus owners like infertility, endometriosis, PCOS/PCOD and many more.
This gave us the opportunity to tap and unleash the potential of commensals to be genetically modified using synthetic biology and help us develop OviCloak. In order to understand how to utilise these bacteria, we first understand the concepts used while designing the genetic circuit that needs to be incorporated into the bacteria during modifications.

Reference

1. Gibson E, Mahdy H. Anatomy, Abdomen and Pelvis, Ovary. [Updated 2021 Jul 26]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK545187/ 
2. Han J, Sadiq NM. Anatomy, Abdomen and Pelvis, Fallopian Tube. [Updated 2021 Jul 31]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK547660/ 
3. Harris EA, Stephens KK, Winuthayanon W. Extracellular Vesicles and the Oviduct Function. Int J Mol Sci. 2020 Nov 5;21(21):8280. doi: 10.3390/ijms21218280. PMID: 33167378; PMCID: PMC7663821.

Gasner A, P A A. Physiology, Uterus. [Updated 2021 May 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557575/ 

4. Rosner J, Samardzic T, Sarao MS. Physiology, Female Reproduction. [Updated 2021 Oct 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK537132/ 
5. Thiyagarajan DK, Basit H, Jeanmonod R. Physiology, Menstrual Cycle. [Updated 2021 Sep 18]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK500020/ 
6. Monis CN, Tetrokalashvili M. Menstrual Cycle Proliferative And Follicular Phase. [Updated 2021 Sep 14]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK542229/ 
7. Holesh JE, Bass AN, Lord M. Physiology, Ovulation. [Updated 2021 May 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK441996/ 
8. Reed BG, Carr BR. The Normal Menstrual Cycle and the Control of Ovulation. [Updated 2018 Aug 5]. In: Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK279054/ 
9. J.C.Harper,Fertilization,Editor(s): Stanley Maloy, Kelly Hughes,Brenner's Encyclopedia of Genetics (Second Edition),Academic Press,2001,Pages 34-35,ISBN 9780080961569,https://doi.org/10.1016/B978-0-12-374984-0.00527-1  (https://www.sciencedirect.com/science/article/pii/B9780123749840005271)
10. AUSTIN C. R. (1956). Cortical granules in hamster eggs. Experimental cell research, 10(2), 533–540. https://doi.org/10.1016/0014-4827(56)90025-8
11. Ducibella, T., Anderson, E., Albertini, D. F., Aalberg, J., & Rangarajan, S. (1988). Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation. Developmental biology, 130(1), 184–197. https://doi.org/10.1016/0012-1606(88)90425-3
12. Wessel, G. M., Brooks, J. M., Green, E., Haley, S., Voronina, E., Wong, J., Zaydfudim, V., & Conner, S. (2001). The biology of cortical granules. International review of cytology, 209, 117–206. https://doi.org/10.1016/s0074-7696(01)09012-x
13. Barros, C., & Yanagimachi, R. (1971). Induction of zona reaction in golden hamster eggs by cortical granule material. Nature, 233(5317), 268–269. https://doi.org/10.1038/233268a0
14. Bleil, J. D., Beall, C. F., & Wassarman, P. M. (1981). Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2. Developmental biology, 86(1), 189–197. https://doi.org/10.1016/0012-1606(81)90329-8
15. Gahlay, G., Gauthier, L., Baibakov, B., Epifano, O., & Dean, J. (2010). Gamete recognition in mice depends on the cleavage status of an egg's zona pellucida protein. Science (New York, N.Y.), 329(5988), 216–219. https://doi.org/10.1126/science.1188178
16. Burkart, A. D., Xiong, B., Baibakov, B., Jiménez-Movilla, M., & Dean, J. (2012). Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. The Journal of cell biology, 197(1), 37–44. https://doi.org/10.1083/jcb.201112094
17. Pires, E.S., Hlavin, C., Macnamara, E., Ishola-Gbenla, K., Doerwaldt, C., Chamberlain, C., Klotz, K., Herr, A.K., Khole, A., Chertihin, O., Curnow, E., Feldman, S.H., Mandal, A., Shetty, J., Flickinger, C. and Herr, J.C. (2013), SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition. Dev. Dyn., 242: 1405-1426.
18. Körschgen, H., Kuske, M., Karmilin, K., Yiallouros, I., Balbach, M., Floehr, J., Wachten, D., Jahnen-Dechent, W., & Stöcker, W. (2017). Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida. Molecular human reproduction, 23(9), 607–616.>https://doi.org/10.1093/molehr/gax040
19. Anna D. Burkart, Bo Xiong, Boris Baibakov, Maria Jiménez-Movilla, Jurrien Dean; Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. J Cell Biol 2 April 2012; 197 (1): 37–44. doi: https://doi.org/10.1083/jcb.201112094
20. Pelzer, E. S., Willner, D., Buttini, M., Hafner, L. M., Theodoropoulos, C., & Huygens, F. (2018). The fallopian tube microbiome: implications for reproductive health. Oncotarget, 9(30), 21541–21551. https://doi.org/10.18632/oncotarget.25059
21. Pelzer, E. S., Willner, D., Huygens, F., Hafner, L. M., Lourie, R., & Buttini, M. (2018). Fallopian tube microbiota: evidence beyond DNA. Future microbiology, 13(12), 1355-1361.

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