Npb 121 MT2

Spermatozoa in the female tract

After insemination, only a tiny selected population of sperm survives long enough to reach the oviduct, capacitate it, bind the oocyte, and fertilize it.

The complete journey after insemination

1. Transverse the cervix and uterus

2. Enter the oviduct and form a functional reservoir

3. Complete capacitation and acquire hyperactive motility

4. Bind to the zona pellucida and undergo the arcosome reaction

5. Fuse with the oocyte and form the male pronucleus

Major sequence of events following deposition of spermatozoa in female tract

1. Immediate transport

• retrograde loss

• Phagocytosis

• Entrance into cervix/uterus

2. Cervix

• “Privileged pathways”

• Removal of non motile sperm

Removal of some abnormalities

3. Uterus

• capacitation initiated

• Phagocytosis

4. oviduct

   • Docking to oviductal cells

   • Capacitation completed

   • Hyperactive motility

5. Fertilization

   • Acrosome reaction

   • Spermatozoon penetrates oocyte

   • Male and female pronuclei form

cow, sheep, rabbit, primates, dog, cat typical deposition or transport feature

Cranial vagina

Pig typical deposition or transport feature

Cervix, semen flows into uterus

Horse typical deposition or transport feature

Cervix/uterus through cervical lumen

Dog typical deposition or transport feature

cranial vagina; third fraction pushes sperm cranially

Boar and stallion typical deposition or transport feature

fractionated ejaculate; viscous fraction reduces loss

Leukocyte Infiltration Helps Prevent Reproductive Tract Infections

The ability of the female to retain viable spermatozoa may influence the fertility of a given mating

Retrograde transport

moves semen caudally toward the exterior

Neutrophils

enter uterine lumen within hours after insemination

Leukocytes

protect tract from infection but also phagocytose sperm

do not perfectly distinguish live from dead sperm

rapid phase sperm transport

sperm can appear in the oviduct within minutes, but many are not functionally viable

driven largely by tract contractions, fluids, and seminal prostaglandins

sustained phase

more biologically important

sperm are released gradually from cervical and uterotubal reservoirs

docking in the oviduct supports survival

The cervix

barrier, filter, and sperm reservoir

estrus produces cervical mucus with different viscosities

Low-viscosity sialomucin in basal crypts creates "privileged pathways”

Viscous sulfomucin helps wash out poorly positioned or non-motile sperm

The cervix therefore selects for motility and anatomical competence

Memorable phrase for cervix

The cervix is not a hallway; it is a selective maze

Capacitation

sperm become fertile inside female tract

exposes molecules required for zona binding

biochemical preparation for fertilization — not simply “sperm aging”

Epididymal maturation

necessary but not sufficient for maximum fertility

Seminal plasma

coats sperm membrane molecules

female tract environment removes or modifies these coatings

decapacitation

Returning sperm to seminal plasma can reverse the process

Oviduct

reservoir, survival niche, and site of final activation

sustained phase in oviduct

sperm enters isthmus and attach to oviductal epithelium aka “docking”

• docking supports sperm survival; without it, sperm survival sharply reduced

what is completed in the oviduct?

capacitation

Motility in the oviduct

changes from progressive linear movement to hyperactive, vigorous, localized movement

Hyperactivation

helps sperm contact and penetrate the oocyte in investments

postcapacitation sequence of events leading to fertilization

hyperactive motility → binding to zona pellucida → acrosomal reaction → penetration of zona pellucida → sperm-oocyte membrane fusion → sperm engulfed → decondensation of sperm nucleus → formation of ,ale pronucleus

Zona pellucida binding

molecular recognition before penetration

Acrosome

sperm membrane over acrosome contains zona-binding regions

capacitation in zona pellucida

exposes binding molecules required for interaction with zona pellucida

ZP3

functions like a receptor-like binding target for sperm

Zona binding

physically attaches sperm and initiates signaling for acrosome reaction

Zona penetration and sperm-oocyte fusion

acrosomal enzymes, including acrosin, digest a local path through the zona pellucida

Sperm enters the perivitelline space and contacts oocyte microvilli

Fusion occurs between oocyte membrane and sperm equatorial segment

The sperm nucleus enters oocyte cytoplasm and begins decondensation

After fusion

1. Cortical reaction

cortical granules release contents into pervitelline space

2. Block to polyspermy

Zona block and/or vitelline block prevent additional sperm entry

3. Pronuclei and syngamy

Sperm nucleus decondenses, male pronucleus forms, the male and female pronuclei fuse

Polyspermy

produces lethal chromosomal imbalance, so the oocyte must rapidly prevent additional sperm entry

Glutathione in oocyte

reduces disulfide cross-links in the sperm nucleus so decondensation can occur

Syngamy

marks formation of zygote and beginning of embryogenesis

cow and mare: anatomy to reproductive management

transcervical insemination targets the uterus

sow: anatomy to reproductive management

insemination with large volume allows flow into uterine horns

bitch and cat: anatomy to reproductive management

insemination used commonly

The correct technique for reproductive management

Respects species anatomy, semen volume, sperm survival, and transport physiology

Fertilization succeeds only when transport, capacitation, zona binding, acrosome reaction, fusion, and pronuclear formation occur in sequence

during reproductive management sperm numbers ____ but sperm function ___

decrease, increase

oogenesis

a pathway from embryo to folliculogenesis and readiness for fertilization

when is the female germ line established?

before birth

where does the oocyte remain

arrested inside the ovarian reserve

folliculogenesis

supports growth, selection, maturation, and ovulation

ovulated secondary oocyte connects to

fertilization

primordial germ cells (PGCs)

embryonic precursors of oocytes and spermatozoa

PCGs in mammals

arise early from embryonic tissues and migrate to the future gonad

PGCs colonize the ____ then they _____ and become _____

future ovary, proliferate, oogonia

• this stage matters because all later female gametes come from this embryonic cell population

Mitosis

diploid primordial germ cells divide several times to form diploid oogonia

• most of these degenerate — only one continues to grow

Growth

food stores are built in oogonia to make a larger diploid cell called a primary oocyte

Meiosis I

• primary oocytes undergo the first meiotic division to produce two haploid cells of very unequal size

• the large cell is the secondary oocyte

• the much smaller cell is the first polar body

• no more changes occur until after ovulation and fertilization by spermatozoa

Meiosis II

Only takes place after fertilization

• secondary oocyte undergoes second meiotic division to form the haploid ovum and another polar body

• First polar body divides to form two more polar bodies

• The three polar bodies simply degenerate and die

Oogenesis step process

1. Oogonium develops from the germinal epithelium overlying the ovary that is a diploid stem-cell also having the self-renewing capacity

2. Oogonium then undergoes mitosis cell division and differentiates into the primary oocytes

3. Primary oocytes undergo meiosis cell division-1, which arrest at diplotene stage in the childhood and puberty onwards, these forms secondary oocyte and one polar body

4. Secondary oocytes further undergo meiosis cell division-2, which arrest at metaphase stage and later forms ootids

5. At last, ootid undergoes process of fertilization and forms a non-motile, large and spherical ovum and second polar body that degenerates

stages of follicular development: primordial follicle

• first stage of follicular development

• formation of primordial follicles occurs during fetal life

• coincident with initiation of meiosis by primary oocyte, the germ cells are enclosed in a single layer of pregranulosa cells — primordial follicle is formed

• Primordial follicles are progressively depleted during reproductive life span of a female individual

• Depletion occurs as a result of two processes

  • atresia

  • entry into the growth phase (i.e. folliculogenesis)

• the mechanism triggering initiation of follicular growth remains largely unknown

Primary Follicle

• The oocyte is surrounded by a single layer of cuboidal granulosa cells

• Formation of the zona pellucida (ZP) begins

• Whether ZP is secreted by the oocyte, surrounding granulosa cells or both remains controversial

• species differences may also exist

Secondary Follicle

• Due to granulosa cell proliferation, the secondary follicle is surronded by several layers of granulosa cell

• Stromal cells near basal lamina become aligned parallel to each other and form the theca interna and theca externa

• Cells of theca interna differentiate into epitheloid cells having all organelles required for steroid secretion

• The cells of theca interna retain spindle-shaped morphology

• Secondary follicles have an independent blood supply for the first time during folliculogenesis (one or two arterioles)

• This blood supply allows the follicle to be exposed to factors circulating in the blood which affect further follicular development

Graafian Follicle / Antral follicle

• in the Graafoam/antral follicle, the fluid-filled cavity has reached its maximal size

• The oocyte is located eccentrically within the antrum

Cumulus oophorus

granulosa cells immediately surrounding the oocyte

Antral granulosa cells

those closest to antral cavity

mural granulosa cells

closest to the basal membrane

• more steroidogenically active than granulosa cells of the cumulus oophorus

What do granulosa cell characteristics depend on?

Granulosa cells exhibit different characteristics depending on location

Preovulatory follicle during final stage of follicular development

becomes a highly vascularized structure

gap junctions

extensive gap junctions exist between granulosa cells and between granulosa cells and oocyte across ZP

LH surge

arrested primary oocyte to ovulation-ready secondary oocyte

• endocrine trigger for final preovulatory transition

Meiotic arrest is released and germinal vesicles breakdown occurs

Meiosis I is completed and the first polar body is extruded

Oocyte proceeds to metaphase II, where it arrests again

Metaphase II secondary oocyte is the cell normally ovulated in most domesticated species

Ootid

Pronuclear stage that occurs after sperm entry, male and female pronuclei can be present within the oocyte cytoplasm

zygote

formed when the pronuclei fuse in syngamy

• single celled

embryo

early developing organism

Blastomeres

cells of a two-celled embryo generated after first cleavage division

cell division process following fertilization

1. fertilization (day 0)

2. Zygote (day 1)

3. 2 cell stage

4. 4 cell stage (day 2)

5. 8 cell stage (day 3)

6. Morula / 10 cell stage

7. Morula / 32 cell stage (day 4)

8. Blastocyst (day 5)

Jelly layer / Zona pellucida

Each egg is surrounded by a jelly layer, composed of glycoproteins (proteins that have sugars struck to them)

• in mammals, this layer is called zona pellucida

• in placental mammals, additional layer of follicular cells surrounds zona pellucida

Vitelline envelope

a second membrane outside of cell’s plasma membrane that separates the Zona pellucida from the egg

Cortical granules

Vesicles that contain enzymes that will degrade proteins that hold vitelline envelope around the plasma membrane

• underneath egg’s plasma membrane

anatomy of sperm

head

• contains tightly packed DNA

flagellar tail

• swimming

many mitochondria

• provide power for sperm movement

Bindin proteins

proteins in plasma membrane of the sperm