National nstitute or asic iology

B

B

Professor

A

osei

Assistant Professor:

HARA, Kenshiro
KITADATE, Yu
NIBB Research Fellow:
NAKAMURA, Yoshiaki*
Technical Staff:
MIZUGUCHI, Hiroko
Postdoctoral Fellow:
NAKAMURA, Yoshiaki
IKAMI, Kanako
TOKUE, Moe
SOKENDAI Graduate Student: IKAMI, Kanako*
TOKUE, Moe*
NONAMI, Yuta
ISHIZAKA, Miho
Technical Assistant:
INADA, Kana
KON, Yayoi
MARUYAMA, Ayumi
Secretary:
KUBOKI, YukoProduction of numerous sperm for a long period in testis is fundamental  for  continuity  of  life  across  generations.  This process  of  spermatogenesis  is  supported  by  the  robust  stem cell system.
The  goal  of  the  Division  of  Germ  Cell  Biology  is  to  fully understand the mammalian sperm stem cell system under the context  of  in vivo tissue  architecture.  We  have  successfully revealed  some  characteristics  of  this  interesting  stem  cell system.  First,  differentiating  germ  cells  that  had  been believed to be irreversibly committed for differentiation still retain  their  self-renewing  potential  and  can  contribute  to stem  cell  pool  maintenance  ( potential  stem  cells ). Secondly,  reversion   from  potential  stem  cells  occurs  at  a higher  frequency  when  testicular  tissue  is  damaged  and regeneration  is  induced.  Thirdly,  the  undifferentiated spermatogonia  populations  including  both  actual   and potential   stem  cells  are  preferentially  localized  near vasculature (vasculature-associated niche). Lastly, stem cells turn over at an unexpectedly frequent and stochastic manner under  a  steady-state  situation  that  continuously  produces sperm  (Nakagawa  et al.,  Dev.  Cell  200   Yoshida  et al., Science  200   Nakagawa et al.,  Science  2010   Klein  et al., Cell Stem Cell 2010). These observations have thrown doubt on  the  prevailing  stem  cell  model,  the  A s   model , 
postulating that  stem cell function is restricted to the singly isolated spermatogonia (As cells).

I. Background: Testicular architecture andsperm stem cell theories

In  mammalian  testis,  a  huge  number  of  sperm  are continuously  produced  over  the  reproduction  period  (1-2 years  in  mice),  which  relies  on  stem  cell  activity.  For decades, the cellular identity and behavior of spermatogenic stem cells has been argued as a fundamental question in the field of spermatogenesis.
The process of spermatogenesis takes place in seminiferous tubules  of  the  testis  (Figure  1).  In  mice,  mitotic  stages  of spermatogenic cells, called  spermatogonia , are divided into undifferentiated   and  differentiating   populations. 0Undifferentiated  spermatogonia   are  found  as  singly isolated cells (As), or syncytia consisting mainly of 2 (Apr) or more  (A al)  cells.  The  formation  of  syncytia  is  due  to incomplete  cell  division,  a  germline-specific  cell  division process  where  cytokinesis  does  not  complete  and  the cytoplasmic  connection  between  daughter  cells  persists  via intercellular bridges.

Figure  1.  Spermatogenesis  in  seminiferous  tubules.  Spermatogenesis progresses  from  the  basal-  to  adluminal  compartment.  Stem  cells  are thought  to  reside  primarily  within  undifferentiated  spermatogonia (brown)  which  are  distributed  sparsely  on  the  basement  membrane. Modified from  ara et al., Cell Stem Cell (201 ). The prevailing stem cell model, called the  As model ,  was proposed  in  1 1,  suggesting  that  stem  cell  activity  is restricted  to  the  population  of  A s  spermatogonia,  while interconnected Apr and Aal syncytia are irreversibly committed to  differentiation  and  no  longer  contribute  to  the  stem  cell pool ( uckins, 1 1  Oakberg, 1 1).
On the other hand, over the last decade, studies from some groups,  including  ours,  have  shown  that  the  population  of undifferentiated spermatogonia can be divided on the basis of their  heterogeneous  gene  expression.  In  undisturbed  steadystate  spermatogenesis,  the  GFR 1   subpopulation  (mainly As, Apr, and fewer Aal) is thought to reside on the top of the hierarchy  As  well  as  maintaining  their  own  population, GFR 1   cells  also  give  rise  to  the  second,  Ngn , subpopulation of undifferentiated spermatogonia (comprised of  more Aal  and  fewer As  and Apr).  Ngn   cells,  although retaining an ability to revert into GFRα1  cells that becomes apparent  during  regeneration  after  tissue  insult,  mostly differentiate  into  differentiating  spermatogonia  and  then meiotic spermatocytes and haploid spermatids in undisturbed situations.
Seminiferous  tubules  represent  a  typical  open   stem  cell niche.  This  makes  a  stark  contrast  to  the  well-investigated closed  niche-supported tissues, such as Drosophila gonads or  mammalian  small  intestine.  The  closed   niche  is  a definitive, specified region of a tissue which typically tethers the  stem  cells,  while  cells  that  exit  this  niche  region  will differentiate. In contrast, seminiferous tubules do not harbor such  a  defined  structure,  but  the  stem  cells  (GFR 1  spermatogonia)  are  scattered  in  the  basal  compartment  with some, but significant, preference to the vasculature between the  tubules.  It  remains  an  important  unsolved  question  how Note  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).

stem  cell  fate  is  regulated  (whether  differentiated  or remaining undifferentiated) in an open niche system. In  201 ,  we  questioned  the  cellular  identity  and  the dynamics  of  the  stem  cells,  as  well  as  the  mechanisms regulating stem cell fate in the recently proposed open niche model, as described below. would  challenge  the  As  model   that  was  proposed  over  0 years ago.

II. Cellular identity and the dynamics ofspermatogenic stem cells

The  aforementioned  studies  illustrate  that  the  dynamics  of GFR 1   spermatogonia  are  an  essential  problem  for  a  full understanding  of  the  identity  and  behavior  of  mouse  sperm stem  cells.  To  address  this,  we  performed  single-cell-level analyses  of  in vivo  dynamics  of  GFR 1   spermatogonia, taking advantage of pulse-labeling and live-imaging studies, combined  with  biophysical  modeling,  in  collaboration  with Dr. Ben Simons of the University of Cambridge, UK.. GFR 1   spermatogonia  lie  scattered  unevenly  on  the basement  membrane  of  seminiferous  tubules  (Figure  ),  but their  local  density  over  a  prolonged  tubule  length  is remarkably  constant  in  adult  mice.  By  pulse-labeling  the GFRα1   spermatogonia  with  GFP,  we  obtained  clonal  fate data of GFRα1  spermatogonia from several days to over a year,  and  showed  that,  although  the  entirety  of  GFR 1  spermatogonia maintained their own population while giving rise  to  differentiating  progeny,  the  individual  GFR 1  spermatogonia  followed  highly  variable  and  intricate  fate behaviors.  These  findings  suggest  that  the  maintenance  of GFRα1+ spermatogonia  is  achieved  by  population asymmetry ,  in  which  balanced  stem  cell  maintenance  and production  of  differentiating  progeny  are  achieved  at  a population-level. We  next  investigated  the  behavior  of  GFR 1  spermatogonia,  by  means  of  in vivo  live-imaging.  From  the numerous  -day-long observations that altogether effectively cover  one  year  of  spermatogonial  behavior,  it  is  indicated that  GFR 1   cells  continually  change  their  morphological states  between  A s,  A pr  and  A al  spermatogonia  through  a combination  of  incomplete  cell  division  (leading  to syncytial  extension)   and  syncytial  fragmentation  (through breakage  of  intercellular  bridges) ,  while  giving  rise  to differentiating  progeny  from  all  of  these  morphological states. The  live-imaging  measurements  indicate  that  the  rates  of incomplete division  and  syncytial fragmentation  may be constant  regardless  of  whether  they  are  syncytia  or As.  We were  then  motivated  to  try  to  capture  these  cells  using  a biophysical  modeling  scheme,  which  essentially  depends only  on  the  rates  of  incomplete  division   and  syncytial fragmentation .  Intriguingly,  the  model  accurately  predicted the  wide  range  of  intricate  clonal  fate  behaviors  in  steadystate over wide time scales from several days to over a year, and  the  recovery  from  tissue  insult  observed  in  laboratory experiments.
The  results  together  indicate  that  the  entirety  of  GFRα1  spermatogonia  (including  both As  and  syncytia)  comprise  a single  equipotent  stem  cell  pool  and  cells  continually interconvert  between As  and  syncytial  states,  while  giving rise  to  Ngn   progeny  (Figure  2).  This  novel  paradigm Figure  2.  A  proposed  stem  cell  dynamics.  GFR 1   spermatogonia comprise  a  single  stem  cell  pool,  in  which  cells  continually  and reversibly  interconvert  between  states  of As, Apr  and Aal  spermatogonia through  incomplete  cell  division  (blue  arrows)  and  syncytial fragmentation  (red  arrows),  while  giving  rise  to  Ngn   cells.  Thus, differentiation-destined  cells  follow  a  series  of  transitions (GFRα1 →Ngn →Kit  downward black arrows) that accompany the extension of syncytial length (rightward black arrows). Ngn  and, to a lesser  extent,  Kit   cells  retain  the  capacity  to  revert  back  into  the GFR 1   compartment  in  a  context-dependent  fashion,  such  as  after tissue  insult  or  during  regeneration  from  damage  etc.  (broken  arrows). Reproduced from  ara et al., Cell Stem Cell (201 ).

II. Undifferentiated spermatogonia show
heterogeneous differentiation competence in
response to retinoic acid.

As  described  above,  seminiferous  tubules  represent  a typical  open  niche  of  stem  cells.  In  particular,  GFRα1 , Ngn ,  and  Kit   spermatogonial  subpopulations  are intermingled  in  the  basal  compartment  (Figure  ).  In addition,  these  are  likely  to  be  ubiquitously  bathed  with retinoic  acid  (RA),  a  strong  differentiation-inducer,  that occurs  periodically  along  with  the  cycle  of  seminiferous epithelium ( .  days per a cycle). So, a fundamental question is  raised   Out  of  the  entire  population  of  undifferentiated spermatogonia  with  stem  cell  potential  on  the  basis  of  the activity  of  post-transplantation  colony  formation,  why  do Ngn  cells preferentially differentiate and why do GFR 1  cells maintain their own population (self-renewal)Figure  .  Localization  of  GFR 1 ,  Ngn ,  and  KIT   spermatogonia.
Representative  images  of  triple  immuno-stained  whole-mount seminiferous tubules of an Ngn3-EGFP mouse. Scale bars   50  m. By  pulse-labeling  the  Ngn   and  GFR 1   populations  by means of the tamoxifen-inducible cre-loxP system, we tested the response of these cells to RA. The results first indicated 1National nstitute or asic iologyBthat  Ngn   cells  rapidly  and  efficiently  differentiated  into Kit  cells in response to RA. In contrast, GFRα1+ cells did not  show  effective  differentiation  induced  by  RA,  but  they generated  the  Ngn   cells  in  an  RA-independent  manner. Thus,  GFR 1   and  Ngn   cells  show  distinctive differentiation competence in response to RA.  We  then  questioned  the  molecular  mechanism  that  caused such  differential  responses  to  RA.  To  this  end,  the transcriptomes  of  GFR 1   and  Ngn   fractions  were compared by gene expression microarray analysis. Among a number  of  genes  involved  in  RA  signal  reception, particularly, retinoic acid receptor gamma (Rarγ), expression was  preferentially  observed  in  Ngn   cells  over  GFR 1  cells. This was also true for the RAR  protein (Figure  ). On the  other  hand,  most  of  the  other  RA-related  genes  showed similar levels of expression. Figure  5.  A  scheme  for  the  fate  allocation  of  undifferentiated spermatogonia  along  the  cycle  of  seminiferous  tubules.  See  text  for details.  Publication List

〔Original paper〕

ara,  K.,  Nakagawa,  T.,  Enomoto,  .,  Suzuki,  M.,  Yamamoto,  M.,  Simons, B.D., and Yoshida, S. (201 ). Mouse spermatogenic stem cells continually  interconvert  between  equipotent  singly  isolated  and syncytial states. Cell Stem Cell 14,  5 - 2.

Figure  .  Expression  of  RAR   in  Ngn ,  but  not  in  GFR 1 , spermatogonia. Representative images of double immuno-stained wholemount  seminiferous  tubules  of  an  Ngn3-EGFP mouse.  Scale  bars    50 m. Modified from Ikami et al., Development 142, 1-11 (2015).

We then addressed the roles of RAR  by ectopic expression in  GFR 1   cells  that  normally  do  not  express  this  gene. Intriguingly,  enforced  expression  of  RAR   provided  the GFR 1  cells with differentiation competence in response to RA.  This  indicates  the  key  role  of  RAR   in  the heterogeneous differentiation competence and the subsequent fate selection of undifferentiated spermatogonia. From  these  results,  heterogeneous  differentiation competence, combined with the periodically but ubiquitously distributed RA, appears to allocate the undifferentiated cells’ fates  for  the  continuity  of  spermatogenesis  (Figure  5).  We propose  that  heterogeneous  differentiation  competence combined  with  spatially  ubiquitous  distribution  of differentiation-inducing  signals  would  be  paradigmatic  for stem cell regulation in an opened niche based model.

2〔Original paper (E-publication ahead of print)〕

Atsumi, Y., Yorinaga, E., Ono,  ., Murayama, I., Nakane,  IY., kegami, K., Ota,  W.,  Arai,  N.,  Tega,  A.,  Iigo,  M.,  Darras,  .M.,  Tsutsui,  K., ayashi, Y., Yoshida, S., and Yoshimura, T. Low temperature-induced circulating  triiodothyronine  accelerates  seasonal  testicular  regression. Endocrinology 201  Nov 1 .