Novel Perspectives

Reproductive

Responsibilities of the

Male Gamete:

M.K Sharma

Metaphase

Anaphase

katanin

microtubuleschromosomeskinetochores

Publications

• Speriolin is a novel human and mouse

sperm centrosome protein. M. Goto, D.A.

O’Brien, E.M. Eddy. Human Reproduction, 2010

• Speriolin is a novel spermatogenic

cell-specific centrosomal protein

associated with the seventh WD motif

of cdc20. M. Goto, E.M. Eddy. JBC, 2004.

Functions of Speriolin • Speriolin,a 53Kd testis-specific protein is :

Localized in the Centrosome in cytoplasm

Binding partner of cdc20 protein Translocates cdc20 to the nucleus Chaperones cdc20 to kinetochores during Meiosis Localizes to Spindle Centrioles during Anaphase in the fertilized Oocyte

Speriolin cdc20 binding

Goto & Eddy. JBC, 2004

Functions of cdc20

• Cell cycle promoting protein:

Localizes to spindle kinetochores Binding partner of MAD1/2 spindle check point proteins in

prometaphase Binds to gamma tubulins of spindle Activation of APC ligase

Immunohistochemical localization of Speriolin in

mouse testis

P-Pachytene spermatocytes. M-meiotic spermatocytes

RS-round spermatids. CS-condensing spermatids.

Meiotic Spermatocyte

Condensing Spermatids

Structure of Centrosome

Schatten & Sun. Molecular Human Reproduction, 2009

• Schematic diagram of a typical mammalian Centrosome composed of two centrioles (mother and daughter centriole) surrounded by a meshwork of pericentriolar material (PCM).

• Both Centrioles are connected through interconnecting fibers. The mother Centriole is distinguished from the daughter Centriole by distal and subdistal appendages.

• The Centrosomal material consists of a fibrous scaffolding lattice with a large amount of coiled-coil Centrosome proteins.

• Centrosome’s three-dimensional architecture is primarily maintained through

specific protein–protein interactions.

• Microtubules are anchored with their minus ends to the Centrosome core structure

• Microtubule growth is regulated by distal plus-end addition of tubulin subunits

Structure of Centrosome

Centrosome Reduction

Manandhar et al. BOR, 2005

• Centrosome reduction during Spermiogenesis.

• Male germ cells possess intact Centrosomes containing Centrioles and Centrosomal proteins until the round spermatid stage (A).

• Microtubules of the distal Centriole extend as axoneme of the spermatid tail (B).

• During Spermiation, g-tubulin and possibly other Centrosomal proteins are disjuncted from the Centrioles and discarded with the residual bodies (B).

• Centrioles are degenerated to various extents in spermatozoa of different species. Rodent and snail spermatozoa lose both Centrioles completely (C),

• Primate spermatozoa retain proximal Centrioles intact but degenerate distal Centrioles partially (D).

• Xenopus and Drosophila spermatozoa possess both Centrioles intact (E).

Centrosome Reduction

Inheritance of Centrioles

Inheritance of Speriolin

In green

Speriolin in mouse and human sperm

mouse

human

Speriolin in fertilized mouse oocyte

In green

Speriolin in mouse male pronucleus

In green

Syngamy in Fertilized Oocyte: Interphase

Schatten & Sun. Molecular Human Reproduction, 2009

Nakamura et al. BOR, 2001

S-Phase

Duplication:

• Centriole

• DNA

Chaperoning cdc20

• Improper Chromosome alignment on the mitotic spindle, disruption of microtubule dynamics, or unattached kinetochores can activate the spindle checkpoint.

• Spindle checkpoint signaling is mediated by the Bub1, Bub3, BubR1, and Mad2 proteins, which all localize to kinetochores.

• These core spindle checkpoint regulators prevent the activator protein cdc20 from binding to APC and therefore protects Securin, a major APC-cdc20 target, from ubiquitin-mediated degradation. As a result, Securin remains bound to Separase, and this prevents cleavage of Scc1 and loss of centromeric Cohesin.

• Spindle checkpoint is relieved at the end of the metaphase by the appropriate biorientation of the sister Chromatids at the metaphase plate. Sensing mechanism involves detecting tension through the Cohesin complex at the kinetochores, created by the pulling of the spindle fibers toward the poles. Mad2 then dissociates from the attached kinetochore, and this allows cdc20 to activate APC and trigger sister Chromatid Segregation.

Martin D. Abeloff, MD; James O. Armitage; John E. Niederhuber; Michael B. Kastan; W. Gillies McKennaAbeloff’s Clinical Oncology: The Spindle Checkpoint

Cdc20 dependent activation of APC

• APC, the Anaphase promoting complex :

Has Ubiquitin ligase activity Expressed in Anaphase Binds to and is activated by cdc20 Ubiquitinates Securin to liberate Separase which disrupts the Cohesins Brings about the Segregation of the Chromosomes

Functions of APC

Speriolin drives Anaphase

Huo et al. BOR, 2004

Speriolin in 2 cell mouse embryo

In green

Implications

• Speriolin is the first reported testis-specific M-protein of the PCM to be inherited from the male gamete

• Putative role is in the regulation of Cell Division in the 1 cell embryo during Anaphase

• Infertility Marker-10% of IVF donors lack sperm associated Speriolin

Reproductive Responsibilities of the Male Gamete

Re-organization of germ cell Chromatin so

as to maintain its Genetic & Epigenetic

integrity

Transfer of the Genetic material to progeny

Transfer of the Centrioles to progeny

Transfer of Speriolin to the progeny

To produce healthy offspring

Thank you

Goto et al. Human Reproduction, 2010

APC drives Meiotic Anaphase in Testes

Segregation of Chromosomes

Division of the 1 cell Preimplantation embryo

Postimplantation Embryogenesis