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Jon M. Oatley · Michael D. Griswold
Editors
The Biology of
Mammalian
Spermatogonia
The Biology of Mammalian Spermatogonia
Jon M. Oatley • Michael D. Griswold
Editors
The Biology
of Mammalian
Spermatogonia
Editors
Jon M. Oatley Michael D. Griswold
Centre for Reproductive Biology School of Molecular Biosciences
School of Molecular Biosciences Washington State University
Washington State University Pullman, WA, USA
Pullman, WA, USA
ISBN 978-1-4939-7503-7 ISBN 978-1-4939-7505-1 (eBook)

https://doi.org/10.1007/978-1-4939-7505-1
Library of Congress Control Number: 2017958850
© Springer Science+Business Media LLC 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
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The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
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Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer Science+Business Media, LLC
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface
Spermatogenesis is a complex and highly efficient process producing millions of

terminally differentiated and specialized sperm every day in males of most mam-
malian species. Indeed, the average man generates roughly 1300 sperm with every
heartbeat, and this level of production is required for fertility. At the foundation of
spermatogenesis are the actions of an undifferentiated spermatogonial population
that consists of spermatogonial stem cell (SSC) and transit amplifying progenitor
pools. Self-renewing divisions of SSCs maintain a reservoir from which progenitor
spermatogonia arise that transiently amplify in number before transitioning from
the undifferentiated type A to differentiating type A1 state under the influence of a
retinoic acid pulse. This transition kick-starts a round of spermatogenesis and the
differentiating spermatogonia gain competence for meiotic initiation. Not only are
these behaviors essential for spermatogenesis during homeostatic conditions but
also during regeneration of the spermatogenic lineage following cytotoxic insult or
transplantation.
The previous two decades has seen an explosion of new information about the
general biology of spermatogonia in mammals and development of methods to iso-
late, culture, genetically modify, and transplant SSCs. Mammalian spermatogonia
have garnered the interest of researchers in the fields of developmental and germ
cell biology for decades because the end product of their activities is sperm that are
the conduit for transmission of a male’s genetic information across generations and
SSCs possess the capacity to regenerate the spermatogenic lineage. These attributes
hold great potential for exploitation to engineer the genetics of the germline directly
which has applications in both improving human health and enhancing the effi-
ciency of animal agriculture.
In this book, we aim to provide a resource of current understandings about vari-
ous aspects of the biology of spermatogonia in mammals. Considering that covering
the entire gamut of all things spermatogonia is a difficult task, specific topics were
selected that we believe provide foundational information that will be useful for
seasoned researchers in the field of germ cell biology as well as investigators enter-
ing the area.
Looking to the future, we predict that the foundational information provided in
this book combined with the advent of new tools and budding interests in the use of
non-rodent mammalian models will produce another major advance in knowledge
regarding the biology of spermatogonia over the next decade. In particular, we
v
vi Preface
anticipate that the core molecular machinery driving different spermatogonial states
in most, if not all, mammals will be described fully, the extrinsic signals emanating
from somatic support cell populations to influence spermatogonial functions will
become fully known, and the capacity to derive long-term cultures of SSCs and
transplant the population to regenerate spermatogenesis and fertility will become a
reality for higher order mammals.
We would like to thank Portia Wong and Dana Bigelow and their staff at Springer
Nature publishing for the patience and assistance in bringing this book to comple-
tion. Also, we would like to express sincere gratitude to all contributors of chapters
in this book for sharing their expertise and insights with the research community.
Pullman, WA, USA Jon M. Oatley

Michael D. Griswold
Contents
Part I Spermatogenesis in Mammals

1 Organization of the Seminiferous Epithelium and the Cycle,
and Morphometric Description of Spermatogonial Subtypes
(Rodents and Primates) �� 3
Dirk G. de Rooij
Part II Postnatal Development of the Spermatogonial Population

2 Transition of Prenatal Prospermatogonia
to Postnatal Spermatogonia�� 23
John R. McCarrey
3 Setting the Stage: The First Round of Spermatogenesis�� 39

Christopher B. Geyer
Part III Spermatogonial Stem Cells

4 Defining the Phenotype and Function of Mammalian
Spermatogonial Stem Cells �� 67
Kazadi N. Mutoji and Brian P. Hermann
5 Regulation of Spermatogonial Stem Cell Maintenance
and Self-Renewal�� 91
Tessa Lord and Jon M. Oatley
Part IV Spermatogonial Differentiation

6 Role of Retinoic Acid Signaling in the Differentiation
of Spermatogonia �� 133
My-Thanh Beedle, Cathryn A. Hogarth, and Michael D. Griswold
7 Gonadotropin and Steroid Hormone Control of Spermatogonial
Differentiation�� 147
Rod T. Mitchell, Laura O’Hara, and Lee B. Smith
vii
viii Contents
Part V Genome Integrity of Spermatogonia

8 Frequency of Human Disease Mutations and Spermatogonial
Stem Cell Function�� 181
Norman Arnheim and Peter Calabrese
9 The Spermatogonial Stem Cell and the Environment�� 205
Tegan S.A. Horan, Caroline V. Sartain, and Patricia A. Hunt
10 Testicular Germ Cell Tumors and Teratomas �� 225
Denise G. Lanza and Jason D. Heaney
Part VI Tools to Study Spermatogonial Biology

11 Transplantation and Culture of Spermatogonial Stem Cells�� 271
Hiroshi Kubota and Ralph L. Brinster
12 In Vitro Differentiation of Spermatogonia�� 301
Takehiko Ogawa
Part VII Therapeutic Potentials and Applications of Spermatogonia

13 Fertility Preservation in Cancer Patients�� 315
Sherin David and Kyle E. Orwig
14 Application of Spermatogonial Transplantation
in Agricultural Animals�� 343
Raquel González, Lin Tang, and Ina Dobrinski
Index�� 379
Contributors
Norman Arnheim Molecular and Computational Biology Program, University of

Southern California, Los Angeles, CA, USA
My-Thanh Beedle School of Molecular Biosciences, Washington State University,
Pullman, WA, USA
Ralph L. Brinster Department of Biomedical Sciences, School of Veterinary
Medicine, University of Pennsylvania, Philadelphia, PA, USA
Peter Calabrese Molecular and Computational Biology Program, University of
Southern California, Los Angeles, CA, USA
Sherin David Department of Obstetrics, Gynecology and Reproductive Sciences,
Molecular Genetics and Developmental Biology Graduate Program, University of
Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, PA,
USA
Ina Dobrinski Department of Comparative Biology and Experimental Medicine,
Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
Christopher B. Geyer Department of Anatomy and Cell Biology at the Brody
School of Medicine and East Carolina Diabetes and Obesity Institute, East Carolina
University, Greenville, NC, USA
Michael D. Griswold School of Molecular Biosciences, Washington State
University, Pullman, WA, USA
Raquel González Department of Comparative Biology and Experimental
Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB,
Canada
Jason D. Heaney Department of Molecular and Human Genetics, Center for
Reproductive Medicine, Dan L. Duncan Cancer Center, Baylor College of Medicine,
Houston, TX, USA
Brian P. Hermann Department of Biology, The University of Texas at San
Antonio, San Antonio, TX, USA
ix
x Contributors
Cathryn A. Hogarth School of Molecular Biosciences, Washington State

Tegan S.A. Horan School of Molecular Biosciences, Washington State University,
Pullman, WA, USA
Patricia A. Hunt School of Molecular Biosciences, Washington State University,
Pullman, WA, USA
Hiroshi Kubota Department of Animal Science, School of Veterinary Medicine,
Kitasato University, Aomori, Japan
Denise G. Lanza Department of Molecular and Human Genetics, Center for
Reproductive Medicine, Dan L. Duncan Cancer Center, Baylor College of Medicine,
Houston, TX, USA
Tessa Lord Centre for Reproductive Biology, School of Molecular Biosciences,
Washington State University, Pullman, WA, USA
John R. McCarrey Department of Biology, University of Texas at San Antonio,
San Antonio, TX, USA
Rod T. Mitchell MRC Centre for Reproductive Health, The Queen’s Medical
Research Institute, University of Edinburgh, Edinburgh, UK
Kazadi N. Mutoji Department of Biology, The University of Texas at San Antonio,
San Antonio, TX, USA
Jon M. Oatley Centre for Reproductive Biology, School of Molecular Biosciences,
Washington State University, Pullman, WA, USA
Takehiko Ogawa Laboratory of Biopharmaceutical and Regenerative Sciences,
Institute of Molecular Medicine and Life Science, Yokohama City University,
Yokohama, Japan
Laura O’Hara MRC Centre for Reproductive Health, The Queen’s Medical
Kyle E. Orwig Department of Obstetrics, Gynecology and Reproductive Sciences,
Molecular Genetics and Developmental Biology Graduate Program, University of
Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, PA,
USA
Dirk G. de Rooij Reproductive Biology Group, Division of Developmental
Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht,
The Netherlands
Center for Reproductive Medicine, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands
Caroline V. Sartain School of Molecular Biosciences, Washington State
Contributors xi
Lee B. Smith MRC Centre for Reproductive Health, The Queen’s Medical
Lin Tang Department of Comparative Biology and Experimental Medicine,
Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
Part I
Spermatogenesis in Mammals
Organization of the Seminiferous
Epithelium and the Cycle, 1
and Morphometric Description
of Spermatogonial Subtypes
(Rodents and Primates)
Dirk G. de Rooij
Abstract
Spermatogenesis encompasses three main cell types: spermatogonia that pro-
liferate, spermatocytes that carry out the process of meiosis, and haploid sper-
matids that develop into sperm. Differentiating spermatogonia are formed at
species-specific intervals, and in each tubule cross-section 4 or 5 generations of
spermatogenic cells can be observed. As the timing of the development of the
germ cells is always similar, specific associations of germ cell types are seen in
tubule cross-sections. Each epithelial area looks similar with regular intervals
and goes through the cell associations in a specific order. This is called the
cycle of the seminiferous epithelium and the cell associations are called stages
(usually 12).
In non-primate mammals, the spermatogonial stem cells (SSCs) are single
cells (As spermatogonia) that divide 2–3 times per epithelial cycle and render
either two new singles (self-renewal) or the daughter cells stay together and form
a pair. The niche for these SSCs likely is that part of the tubule basal lamina that
borders on interstitial venules and arterioles. The pairs divide further into chains
of spermatogonia that differentiate during epithelial stage VIII. The organization
of the epithelium heavily depends on a peak in retinoic acid levels during stages
VIII-IX that both induces spermatogonia to differentiate and preleptotene sper-
matocytes to enter meiotic prophase. Surprisingly, germ cells can develop nor-
mally while in abnormal epithelial stages, the strict epithelial organization is not
required for qualitatively normal spermatogenesis.
D.G. de Rooij, Ph.D. (*)

Reproductive Biology Group, Division of Developmental Biology, Department of Biology,
Faculty of Science, Utrecht University, Padualaan 8, Utrecht, The Netherlands
Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
e-mail: d.g.derooij@uu.nl
© Springer Science+Business Media LLC 2017 3

J.M. Oatley, M.D. Griswold (eds.), The Biology of Mammalian Spermatogonia,
https://doi.org/10.1007/978-1-4939-7505-1_1
4 D.G. de Rooij
The present knowledge on primate SSCs and spermatogonial multiplication is

discussed but does not yet allow clear conclusions.
Keywords
Spermatogonia • Cycle of the seminiferous epithelium • Stages of the seminifer-
ous epithelial cycle • Wave of the seminiferous epithelium • Spermatogonial
kinetics • Spermatogonial stem cells • Stem cell niche • Retinoic acid
1.1 Introduction
Spermatogenesis takes place in the seminiferous epithelium that lines the seminifer-
ous tubules in the testis. The seminiferous tubules begin and end in the rete testis.
Spermatozoa when released into the lumen of the tubules are transported to the rete
testis and subsequently proceed through the efferent ducts to the epididymis.
Besides the various types of germ cells, the seminiferous epithelium consists of the
somatic Sertoli cells that produce many factors that are needed at various develop-
mental steps during the spermatogenic process (for reviews see Griswold 2015).
During spermatogenesis, the germ cells have to pass through many different dif-
ferentiation steps to develop into the highly specialized spermatozoa (Russell et al.
1990). The whole process can be subdivided into three main parts. First, there is a
phase of cell proliferation which in many species involves about ten subsequent
divisions from stem cells up to the formation of the next major cell type, called
spermatocytes. Second, the spermatocytes carry out meiosis, a process that encom-
passes a recombination of hereditary characteristics and a reduction in the number
of chromosomes by way of the two consecutive meiotic divisions. Third, through
the meiotic divisions, spermatocytes give rise to haploid spermatids that during a
long, intricate process transform and develop into spermatozoa that leave the testis.
This book will specifically deal with the first of the three main parts of spermato-
genesis, the proliferation phase. This part is carried out by a cell type called sper-
matogonia, among which there are the spermatogonial stem cells (SSCs) that ensure
that spermatogenesis will be maintained throughout the lifespan of mammals.
An important particularity of spermatogenesis is that it is very strictly organized,
both with respect to the duration of the whole process as well as to the localization
of the subsequent cell types within the seminiferous epithelium. This organization
determines the length of the period of time available for spermatogonial prolifera-
tion and even the timing of some crucial differentiation steps. This chapter describes
the organization of the seminiferous epithelium, the various spermatogonial cell
types, and how their cell kinetic behavior fits in the organization of the epithelium.
The important role of spermatogonial stem cells (SSCs) will be highlighted, as well
as their supposed localization in the epithelium, called niche.
1 Organization of the Seminiferous Epithelium and the Cycle, and Morphometric… 5
1.2 The Organization of the Seminiferous Epithelium
As described above, spermatogenesis encompasses a great many subsequent types

of germ cells. Nevertheless, looking at a cross-section of a seminiferous tubule, one
does not see a random mixture of these cell types. There are multiple levels of orga-
nization that make the appearance of the seminiferous epithelium less confusing
(Fig. 1.1).
1.2.1 The Clonal Organization of the Seminiferous Epithelium
As discussed below, the prevalent opinion is that SSCs are single cells. However,
when SSCs differentiate, their daughter cells will stay connected by an intercellular
Fig. 1.1 Cross-section through a mouse seminiferous tubule showing the intricate organization of
the epithelium. This cross-section is in stage VI of the cycle of the seminiferous epithelium. In the
outermost layer (1) there are spermatogonia. In stage VI one finds late differentiating type B sper-
matogonia (some are indicated with B) which are numerous. In this stage, B spermatogonia go
through a division to form preleptotene spermatocytes. Indeed, some of the B spermatogonia can
be seen dividing (m) and some newly formed preleptotenes (preL) are already present. Intermingled
with the B spermatogonia are As,pr,al spermatogonia. These cells are few in number (arrows) and
largely consist of Aal spermatogonia that will become differentiating type A1 spermatogonia in
stage VIII. One layer up into the tubule (2) one can find pachytene spermatocytes which originate
from B spermatogonia present in this area one epithelial cycle earlier (8.6 days). In layer 3 round
spermatids can be found that (per definition) are in step 6 of their development. In layer 4 there are
elongated spermatids that have already moved close to the lumen into which they will be shed dur-
ing stage VIII. In total there are five generations of spermatogenic cells each differing 8.6 days of
development from each other. Note that within each layer, each generation of germ cells is at the
same moment of its development
6 D.G. de Rooij
bridge and form a pair. In subsequent divisions also, intercellular bridges remain
between the daughter cells because of which larger and larger clones of intercon-
nected spermatogonia are formed. The cells composing these syncytial clones will
behave closely similar as regulating substances can pass freely through the bridges
throughout the clone. Therefore one will always see large cohorts of germ cells
being at the same step of development and with respect to spermatogonia even in the
same phase of the cell cycle (Dym and Fawcett 1971). On top of the presence of
clones of synchronized cells there is also synchronization between neighboring
clones which makes the cohorts of germ cells at the same step of development even
larger (Lok and de Rooij 1983a). So, in a tubule cross-section one does not see a
mixture of many cell types in different phases of development but a restricted num-
ber of groups of similar looking cells (Fig. 1.1).
1.2.2 Timing of Germ Cell Development
The timing of the developmental steps germ cells go through is rather constant. There
are species differences but for each species the total duration of the spermatogenic
process, from stem cell to spermatozoa released from the epithelium, is always the
same. Also, the duration of each step of development of the germ cells is fixed. In
addition to the timing of the developmental steps the germ cells make, in any particu-
lar area of the seminiferous epithelium the time interval with which spermatogonia
differentiate and go on their way to become spermatozoa is also fixed. For example,
in the mouse a cohort of spermatogonia differentiates every 8.6 days. However, the
development from newly formed differentiating spermatogonia to spermatozoa
released into the tubule lumen takes about 34 days. Consequently, new cohorts of
differentiating spermatogonia will be formed before the descendants of the previous
cohorts have left the epithelium as spermatozoa. Therefore, in each area of the semi-
niferous epithelium 4–5 generations of germ cells can be observed and in the mouse
each of the successive generations differ by 8.6 days of development. As a result of
the fixed duration of all developmental steps and the interval with which differentiat-
ing spermatogonia are formed, one will always find the same associations of types of
germ cells together Fig. 1.2). In the mouse, each area of the epithelium goes through
a similar sequence of combinations of cell types every 8.6 days and will look similar
with 8.6 day intervals. This is called the cycle of the seminiferous epithelium that can
be observed in all mammals. While in the mouse the cycle takes 8.6 days (Oakberg
1956b), its duration differs for each species (Hess and Renato de Franca 2008). For
example, in the rat the epithelial cycle takes 12.8–13.0 days (Hilscher et al. 1969;
Huckins 1971a) and 16.0 days in the human (Heller and Clermont 1963).
1.2.3 Stages of the Cycle of the Seminiferous Epithelium
As in spermatogenesis one always sees the same associations of types of germ cells,
it follows that when in a particular area of the epithelium one can identify one of the
cell types, one can predict the presence of the other types of germ cells in the same
Fig. 1.2 Diagram of the cycle of the seminiferous epithelium in the mouse. The cycle is subdi-
vided into 12 stages that are generally indicated by roman numerals. The stages are defined by the
first 12 steps of spermatid development. The cell associations that can be seen in a stage are always
similar. Therefore, when in a particular area the epithelial stage is defined looking at spermatid
morphology, one will know which germ cell types will be present too. The As, Apr, and Aal sper-
matogonia are always present and proliferate at random but are most active in stages X to II. The
As spermatogonia can both self-renew and produce Apr spermatogonia. Most of the Aal spermato-
gonia differentiate into A1 spermatogonia without division in stage VIII. Abbreviations: As Asingle
spermatogonia (spermatogonial stem cells), Apr Apaired, Aal Aaligned, preL preleptotene spermatocytes,
L leptotene spermatocytes, Z zygotene spermatocytes, P pachytene spermatocytes, D diplotene
spermatocytes, 1 to 16 steps in the development of the spermatids
tubule area. Therefore, for many species the cell associations have been described
and they have been called the stages of the cycle of the seminiferous epithelium.
The most popular way to subdivide the epithelial cycle into stages is by using the
clearly identifiable steps in the development of the acrosome in spermatids which
can be easily made visible by staining using the periodic acid—Schiff reaction
(Leblond and Clermont 1952b) (Fig. 1.2). This was first done for the rat (Clermont
and Perey 1957; Leblond and Clermont 1952a) in which the cycle was subdivided
in 14 stages, using the first 14 steps of the spermatid development to mark the vari-
ous stages. For the mouse, Oakberg described 12 stages (Oakberg 1956a) and this
number of stages has remained the most popular for other species for which semi-
niferous epithelial stages have been described. Unfortunately, the periodic acid
Schiff (PAS) reaction does not work well on human spermatids and therefore, until
recently the human epithelial cycle could only be subdivided into 6, very long,
stages (Clermont 1963). This made it very difficult to compare findings on human
spermatogenesis with those on experimental animals. Fortunately, the cycle of the
human seminiferous epithelium has now also been subdivided in 12 stages, using
8 D.G. de Rooij
Fig. 1.3 Seminiferous tubule showing part of the wave of the seminiferous epithelium. The stages
of the cycle of the seminiferous epithelium follow each other in order along the length of a semi-
niferous tubule but the order of stages can also reverse. In this tubule the wave goes from stage I to
stage V and then the order reverses. Because of the wave one can also see the synchronous devel-
opment of the differentiating type spermatogonia in order. In stage I, one will find A4 spermatogo-
nia that divide into In spermatogonia in stage II which in turn will divide into B spermatogonia in
stage IV. It is even known in which epithelial stage a particular type of differentiating spermatogo-
nia goes through the various phases of the cell cycle, for example B spermatogonia are in S phase
during stage V. The As, Apr, and Aal spermatogonia cycle at random during the epithelial cycle but
are most active during stages X to II (Lok and de Rooij 1983b)
immunohistochemical staining for acrosin in the acrosomes, which gives a rather

comparable result as PAS staining in the mouse and other mammals (Muciaccia
et al. 2013).
1.2.4 Wave of the Seminiferous Epithelium
In most mammals, but not in human and some other primates, there is a phenome-
non called the wave of the seminiferous epithelium (Perey et al. 1961; Johnson
1994). Studying the spermatogenic process in whole mounts of seminiferous tubules
one can see that each epithelial stage occupies a certain length of tubule and that the
stages follow each other in order along the length of the tubules (Fig. 1.3). Although
the order of the stages sometimes reverses, this is of great help when studying sper-
matogenesis in whole mounts. A further great advantage of studying whole mounts
is that one can always observe the whole of the nuclei of the spermatogonia instead
of parts of them, as in sections, enabling a much better distinction between the vari-
ous spermatogonial cell types (Clermont and Bustos-Obregon 1968; Huckins
1971c; de Rooij 1973). In addition, one can study the topographical arrangement of
the spermatogonia. The latter has been proven essential for a proper understanding
of the spermatogonial compartment. Unfortunately, studying tubule whole mounts
of the human does hardly provide these advantages as in the human, tubule areas in
the same epithelial stage are very small. So small that even a cross-section of a
human seminiferous tubule shows multiple epithelial stages (Clermont 1963). The
same situation has been found in several other primate species (Wistuba et al. 2003).
1.3 Spermatogonial Cell Types
1.3.1 Non-primate Mammals
In the seminiferous epithelium, SSCs are at the start of the spermatogenic process
and produce both new SSCs and spermatogonia that enter the differentiation path-
way. Most of our knowledge on SSCs and spermatogonia comes from studies on
rodents. In the prevailing scheme of spermatogonial multiplication and stem cell
renewal, the SSCs are single spermatogonia (Huckins 1971c; Oakberg 1971; de
Rooij 1973). These single spermatogonia are called As spermatogonia. Upon divi-
sion, the As spermatogonia divide and either produce two new single cells that
migrate away from each other or the daughter cells can stay together, connected by
an intercellular bridge, forming a pair of cells called Apr spermatogonia. At subse-
quent divisions the daughter cells will always remain connected by intercellular
bridges because of which clones of increasing size will be formed at each division.
Subsequently, chains of 4, 8 and 16 cells are formed that are called Aal spermatogo-
nia. The scheme of spermatogonial multiplication and stem cell renewal is still
debated (de Rooij and Griswold 2012). The opinions varying from stem cell renewal
taking place by way of fragmentation of chains of Aal spermatogonia (Nakagawa
et al. 2007, 2010) to only few of the As spermatogonia having stem cell properties
(Chan et al. 2014; Oatley et al. 2011). The virtues of and problems with these alter-
native schemes are discussed in another chapter of this book.
The chains of Aal spermatogonia can differentiate, without an intervening divi-
sion, into so-called A1 spermatogonia, the first generation of a cell type called dif-
ferentiating spermatogonia. The differentiating spermatogonia comprise 6
generations of cells in mouse and rat which are called: A1, A2, A3, A4, In
(Intermediate) and B spermatogonia. The B spermatogonia divide into spermato-
cytes called preleptotene spermatocytes at first and subsequently, when they enter
meiotic prophase, leptotene spermatocytes. In all, in rodents there are about ten
divisions in between the SSCs and the formation of spermatocytes (de Rooij and
Russell 2000).
1.3.2 Primates
While one would not expect big differences between primate and non-primate
mammals in a crucially important process as spermatogenesis, this seems neverthe-
less to be the case. In primates, a type of spermatogonia is distinguished that is
totally missing in non-primates. These are the so-called Adark spermatogonia (Ad). Ad
spermatogonia are characterized by nuclei that stain darkly with hematoxylin. In
addition, Ad spermatogonia show a rarefaction area in their nuclei. In human this
rarefaction zone consists of a vacuole like space in the middle of the nuclei and in
monkeys the rarefaction zone is at the circumference of the nuclei in between the
nuclear membrane and the chromatin. Besides Ad, in primates Apale (Ap) spermato-
gonia can be distinguished that stain more lightly with hematoxylin and do not show
10 D.G. de Rooij
Fig. 1.4 Spermatogonial multiplication and stem cell renewal in primates. It is not yet clear how
these events proceed. In primate spermatogenesis there are Adark spermatogonia that possibly are
the ultimate stem cells that very slowly proliferate and replenish Apale spermatogonia that may not
be able to fully maintain themselves. Alternatively, the Adark spermatogonia are set aside by Apale
spermatogonia and function as reserve stem cells and are normally quiescent but become active
Apale spermatogonia again when locally the Apale population becomes depleted, as after irradiation
a rarefaction zone in the nuclei. Unfortunately, the distinction between Ap and Ad

spermatogonia is not straightforward. Spermatogonia with a different or intermedi-
ate morphology between Ap and Ad, have also been described (Rowley et al. 1971;
Fouquet and Dadoune 1986; Simorangkir et al. 2005). Besides Ap and Ad spermato-
gonia, in monkeys and human, varying numbers of generations of B spermatogonia
have been described.
In general, Ap and Ad spermatogonia together are supposed to be comparable to
the category of As, Apr and Aal (As,pr,al) spermatogonia in rodents, (review Hermann
et al. 2010) (Fig. 1.4). A peculiar aspect of primate spermatogenesis is that the den-
sity of the spermatogonia is very high. This precludes a study of clonal sizes of Ap
and Ad spermatogonia as the clones are often too close to each other to allow one to
distinguish to which clone a particular cell belongs. Therefore, devising a scheme of
spermatogonial multiplication and stem cell renewal for primate spermatogenesis
has proven to be difficult. The Ad spermatogonia do not incorporate 3H-thymidine
and BrdU and also not become labeled one epithelial cycle after administration of
either of the two S phase markers (Clermont 1969; Clermont and Antar 1973; Kluin
et al. 1983; Fouquet and Dadoune 1986; Schlatt and Weinbauer 1994; Ehmcke et al.
2005a, b; Simorangkir et al. 2009). However, Ad spermatogonia can start prolifera-
tion again. During the first epithelial cycle after irradiation, the Ap spermatogonia
become severely reduced in numbers because of the irradiation damage, killing
these cells when they try to divide. Then the Ad spermatogonia become activated
and become Ap spermatogonia (van Alphen et al. 1988). Therefore, the Ad spermato-
gonia are considered to be reserve stem cells that only become active when the Ap
spermatogonia in a particular area fail. Alternatively, the Ad spermatogonia may be
the real stem cells with a very long cell cycle, slowly giving rise to Ap with a
restricted self-renewal capacity in order to maintain the numbers of these cells
(Clermont 1966; Hermann et al. 2010; Clermont and Leblond 1959). The presence
of long-cycling SSCs has been suggested to occur in the rat (Huckins 1971b), but
these findings could not be confirmed in the Chinese hamster (Lok et al. 1984).
Clearly, more work will have to be carried out to understand how spermatogonial
multiplication and stem cell renewal in primates is carried out. Too little is known
yet about the nature and the behavior of both Ap and Ad spermatogonia.
1.4 Cell Kinetics
1.4.1 Non-primates
The As, Apr, and Aal spermatogonia (As,pr,al) are often called undifferentiated sper-
matogonia but as discussed previously (de Rooij and Russell 2000), this is not a
good term as most of these cells (Apr and Aal) are on the differentiation pathway. The
clones of As,pr,al spermatogonia divide randomly throughout the epithelial stages.
However, the overall proliferative activity of As,pr,al spermatogonia varies with the
stages of the epithelial cycle and these spermatogonia are most active in epithelial
stages X to II. From stage II onwards, the proliferative activity decreases, especially
that of Apr and Aal spermatogonia (Lok and de Rooij 1983a, b). In the mouse and
Chinese hamster, As, Apr and Aal spermatogonia go on average through 2–3 divisions
each epithelial cycle (Lok et al. 1983; Lok and de Rooij 1983b; Tegelenbosch and
de Rooij 1993).
In stages VII and VIII, the Aal spermatogonia that were quiescent since stage II,
differentiate into A1 spermatogonia, the first of the series of 6 generations of dif-
ferentiating type spermatogonia. The differentiating spermatogonia have a different
behavior compared to the As,pr,al spermatogonia. Their cell cycle is shorter and
importantly, in contrast to the As,pr,al spermatogonia, the clones of these cells behave
in a synchronized manner. Neighboring clones enter the cell cycle at about the same
time (Huckins 1971a; Lok and de Rooij 1983a). The synchronization of neighbor-
ing clones is such that all clones of a generation of differentiating spermatogonia
present in a particular area will traverse the cell cycle in unison. For example, in
whole mounts of seminiferous tubules extended areas can be seen in which all dif-
ferentiating spermatogonia are in S phase. Therefore, it is highly unlikely that clones
of differentiating spermatogonia can skip a division.
1.4.2 Primates
Comparing spermatogonial multiplication in primates and non-primate mammals,

one has to take into account the completely different ratio between the numbers of
undifferentiated (As,pr,al in non-primates and Ap + Ad in primates) and differentiating
type spermatogonia. In the mouse, there are 7.6 times more differentiating type
spermatogonia per Sertoli cell than As,pr,al spermatogonia (Tegelenbosch and de
Rooij 1993). In contrast, in Macaca fascicularis there are 0.9 times less differentiat-
ing spermatogonia than Ap + Ad spermatogonia (Zhengwei et al. 1997) and for the
12 D.G. de Rooij
human it can be calculated from data from Rowley (1971) that there are even 0.3
times less differentiating spermatogonia than Ap + Ad spermatogonia. Nevertheless,
the numbers of differentiating spermatogonia per Sertoli cell, produced in mice and
primates do not differ all that much. In the mouse there are 0.83 differentiating
spermatogonia per Sertoli cell, in Macaca fascicularis it is 0.43 and in the human
0.31.
What it comes down to is that in rodents the relatively few As,pr,al spermatogonia
go through many divisions to produce the required numbers of differentiating sper-
matogonia while in primates there are a great many Ap and Ad spermatogonia, the Ap
of which divide once or twice every epithelial cycle, or even less, to produce an only
somewhat smaller amount of differentiating spermatogonia.
1.5 Regulation of the Epithelial Cycle
As described above, the proliferative activity of the As,pr,al spermatogonia strongly

depends on the stages of the epithelial cycle. In the mouse, every 8.6 days a new
cohort of Aal spermatogonia is induced to differentiate into A1 spermatogonia. This
event takes place in stages VII and VIII of the epithelial cycle (Schrans-Stassen
et al. 1999) and these newly formed A1 spermatogonia will divide into A2 sper-
matogonia in stage IX. The subsequent divisions will also take place in specific
stages and ultimately B spermatogonia are formed. In stage VI, these B spermato-
gonia will divide to become preleptotene spermatocytes that will enter meiotic pro-
phase at the end of stage VIII. How can all spermatogenic events be timed so
accurately (de Rooij and Russell 2000)?
In recent years, it has become clear that retinoic acid plays an important role in
the regulation of epithelial cycle events. It was already known that in case of vitamin
A deficiency (VAD), in the mouse and rat, spermatogenesis becomes arrested
(Mitranond et al. 1979; Unni et al. 1983). This arrest has been localized to the dif-
ferentiation step of Aal into A1 spermatogonia and in the VAD rat preleptotene sper-
matocytes are unable to enter meiotic prophase (van Pelt and de Rooij 1990a, b;
Ismail et al. 1990). It has been established that retinoic acid (RA) is necessary for
preleptotene spermatocytes to enter meiotic prophase (Baltus et al. 2006; Anderson
et al. 2008). Finally, RA has also been implicated in the release of spermatozoa dur-
ing spermiation (Vernet et al. 2006, 2008). So, RA is required at three essential steps
in spermatogenesis, the differentiation of Aal into A1 spermatogonia, the entrance of
preleptotene spermatocytes into meiotic prophase and spermiation. All three steps
take place in stage VIII of the epithelial cycle. Importantly, it has recently been
established that RA levels in the seminiferous epithelium peak at stages VIII and IX
(Hogarth et al. 2015). Apparently, it is this peak in RA levels that induces spermato-
gonial differentiation, entry of preleptotene spermatocytes into meiotic prophase
and spermiation.
A recent study into the effects of RA administration on the spermatogenic pro-
cess has revealed some aspects of how the strict organization of the seminiferous
epithelium is brought about (Endo et al. 2015). Injection of RA has a very drastic
effect on the quiescent Aal spermatogonia present in stages II to VI at the time of
administration. Normally these cells will differentiate into A1 spermatogonia dur-
ing stage VIII but after injection of RA they do so within 24 h despite the fact that
the surrounding epithelium is in stages II to VI. Apparently, the Aal in stages II to VI
are already competent to differentiate into A1 spermatogonia but normally have to
wait for the higher RA levels in stage VIII to do so. Furthermore, the administration
of RA also induces preleptotenes to enter S phase, and subsequently meiotic pro-
phase, at an earlier time than they normally do (Endo et al. 2015). Apparently,
preleptotenes too are competent to take the next step in their development at an
earlier time point than stage VIII. It can be concluded that the strict organization of
the epithelium with its ever similar cell associations (stages) is at least for a large
part due to the fact that both Aal spermatogonia and preleptotene spermatocytes are
already competent to take their next developmental steps before stage VIII and the
RA peak then causes these cells to simultaneously proceed their development. It is
not known whether RA has a synchronizing effect on the development of pachytene
spermatocytes and the onset of the elongation of step 8 spermatids in stage VIII. As
mentioned earlier spermiation is dependent on RA levels (Vernet et al. 2006, 2008)
but it is not known whether spermatids are already competent to be spermiated
before stage VIII and are also waiting for the RA peak to occur.
The very strict organization of the seminiferous epithelium has always been
interpreted as a requirement for a proper regulation of the spermatogenic process.
For example, Sertoli cells will then be able to timely supply the various types of
germ cells with necessary factors to take their next developmental step. The study
by Endo et al. has thoroughly undermined such ideas (Endo et al. 2015). As men-
tioned above, RA administration drives Aal spermatogonia in stages II to VI to a
prescheduled differentiation into A1 spermatogonia. Surprisingly, these A1 sper-
matogonia subsequently develop into A2, A3, A4, In and B spermatogonia at a
normal pace and preleptotene spermatocytes can be seen in stages II to VI one
epithelial cycle later. These preleptotenes then need RA to enter meiotic prophase
and subsequently proceed through meiotic prophase normally. These observations
strongly suggest that germ cells do not need a stage-specific guidance for their
development, for example through the secretion of stage-specific factors by Sertoli
cells. In support of that notion, rat germ cells transplanted into mouse testes
develop at the same rate as in the rat testis despite the fact that they are surrounded
by mouse somatic cells (Franca et al. 1998). Apparently, for their development the
rat germ cells do not depend on guidance from the environment. In conclusion,
although the seminiferous epithelium is highly organized, this organization does
not seem required for a proper development of the germ cells. It may just be a
consequence of the dependency of several crucial developmental steps on RA and
the fact that there is only one peak in RA levels every epithelial cycle. Germ cells
can develop perfectly well in inappropriate stages though it cannot be excluded
that this may occur less efficiently.
14 D.G. de Rooij
1.6 The Spermatogonial Stem Cell Niche
In all tissues in which the functioning cells have a finite lifespan or, as in the testis,
will leave the tissue, new cells will have to be constantly produced to replenish the
lost cells. In such tissues, there will be stem cells to make sure that the tissue is
maintained. Stem cells are able to both renew themselves and to produce cells that
will become the functional cells of the tissue. In this way, the tissue will be main-
tained throughout life. It has been generally found that stem cells are not distributed
at random in a tissue. Instead, for example in the intestine, hemopoiesis and liver
they occupy specific parts in which surrounding cells provide an environment that
stimulates stem cell self-renewal, (Schofield 1978; Stange and Clevers 2013;
Snippert et al. 2010; Ugarte and Forsberg 2013; Smith and Calvi 2013; Kordes and
Haussinger 2013). Such an area is called a stem cell niche. Outside of the stem cell
niche the environment will induce differentiation of those daughter cells of the stem
cells that venture out of the niche. In this way the tissue secures its necessary com-
plement of stem cells and also takes care that those daughter cells of stem cells that
spill out of the niche will differentiate. In the niche, stem cells will not get lost
because differentiation of these cells is suppressed and on the other hand they will
not be able to propagate in numbers and form a tumor outside of the niche because
they will be forced to differentiate.
In the testis, all spermatogonia including the SSCs are on the basal lamina of the
seminiferous tubules together with the somatic Sertoli cells. Sertoli cells play an
important role in the functioning of the stem cell niche (de Rooij 2009, 2015).
Importantly, it cannot be that all Sertoli cells serve as a SSC niche environment as
in that case no differentiation would take place. For any tissue it is of equal impor-
tance that half of the cells produced by the stem cells differentiate because other-
wise the tissue would keep growing or a tumor would even be formed.
For the testis, it has been shown that the clones of As,pr,al spermatogonia are not
distributed at random over the tubule basal lamina. Several reports show that those
areas of the seminiferous tubules that border on the interstitial tissue and in particu-
lar on venules and arterioles in the interstitial tissue, contain most As,pr,al spermato-
gonia (Chiarini-Garcia et al. 2001, 2003; Yoshida et al. 2007b; Shetty and Meistrich
2007; de Rooij 2009). The numbers of clones of As,pr,al spermatogonia are highest
in these areas suggesting that they originate in these areas and that the SSCs should
reside there too. Unfortunately, it is very difficult to study the niche area directly.
When preparing whole-mounts of seminiferous tubules the blood vessels are
removed because it is difficult to see the spermatogonia under the blood vessels.
Furthermore, in the live imaging system of the Yoshida group the niche area is in a
plane perpendicular to the plane of observation making it impossible to follow
spermatogonial behavior under the interstitial blood vessels. Therefore, a computer
model of SSC behavior has been established in which the SSC niche is supposed
to be directly opposite to the arterioles and venules in the interstitial tissue (de
Rooij 2015; de Rooij and van Beek 2013). This model is based on parameters con-
sistent with the many studies on spermatogonial and SSC proliferation. Within the
niche the chance of self-renewal of the SSCs is very high, while out the niche the
Fig. 1.5 The spermatogonial stem cell (SSC) niche is located in those areas of the tubule basal
lamina that borders on arterioles and venules in the interstitial tissue (Yoshida et al. 2007a). Here
many As spermatogonia are localized. In the niche there is primarily self-renewal and daughter
cells will migrate away from each other. Those daughter cells that migrate out of the niche will at
their next division form a pair of Apr spermatogonia that eventually will become spermatozoa
chance of differentiation of SSCs is high. The SSCs are supposed to be single cells
and when they go through a self-renewing division the daughter cells migrate away
from each other. During this migration both daughter cells may stay in the niche
and will not differentiate. However, in other cases one or both daughter cells may
end up outside of the niche and at their next division differentiate and become Apr
spermatogonia (Fig. 1.5). Indeed, in the live-imaging system of the Yoshida group
which enables one to follow the behavior of clones of As,pr,al spermatogonia outside
of the niche, virtually all As spermatogonia are seen to form Apr spermatogonia
after division (Hara et al. 2014). The computer model renders a stable situation in
which the virtual spermatogenesis continues for more than one and a half year of a
mouse life without the formation of a virtual tumor or SSC depletion (de Rooij and
van Beek 2013).
Intriguingly, the situation may be more complicated than described above. In cell
counts in whole mounts of mouse seminiferous tubules it was established that in the
3H1 mouse 10.6% of the undifferentiated spermatogonia are As (Tegelenbosch and
de Rooij 1993). However, the only cells in the seminiferous epithelium capable to
repopulate a recipient mouse testis after transplantation express Id4 (inhibitor of
differentiation 4) (Chan et al. 2014). These Id4+ spermatogonia are single cells and
they comprise 1.9% of the Plzf+ spermatogonia, representing the total of undiffer-
entiated spermatogonia. This means that there are many more As spermatogonia
than there are SSCs capable of colonizing a recipient mouse testis. Moreover, the
Id4+ As spermatogonia are not localized to the blood vessels in the interstitial tissue.
Chan et al. (2014) suggest that the colonizing SSCs are a kind of stem cells that
should be placed before the other As spermatogonia in the scheme of spermatogo-
nial multiplication and stem cell renewal in the mouse. A hierarchy of stem cells is
suggested in which the population of As spermatogonia does not fully maintain
itself and needs As spermatogonia produced by ID4+ cells. Also, the Id4+ As
16 D.G. de Rooij
spermatogonia probably have another type of niche in a different location as they

are not localized close to the interstitial blood vessels. Clearly, further work will be
needed to get an understanding of the role of the Id4+ As spermatogonia in the nor-
mal seminiferous epithelium and of how the rodent spermatogonial stem cell com-
partment operates during steady state kinetics.
In conclusion, like in all other tissues, SSCs will not be spread over the seminif-
erous epithelium at random. There must be specific areas in which their chance of
self-renewal is strongly enhanced while outside of the niche the differentiation of
SSCs is strongly promoted. In this way, the sustained presence of SSCs in their
niche is guaranteed while on the other hand any surplus SSCs that spill out of the
niche will differentiate and eventually leave the epithelium as sperm. As to the
localization of the niche, present data indicate that for most of the As spermatogonia
it is located in areas opposing arterioles and venules in the interstitial tissue. In addi-
tion, there are relatively rare Id4+ As spermatogonia that are capable of colonizing
a recipient mouse testis and likely have another type of niche and produce a kind of
transient As spermatogonia to restock possible deficits in As numbers. As discussed
elsewhere, Sertoli cells have a very important role in SSC maintenance and differ-
entiation, producing mainly self-renewal factors within the niche and differentiation
promoting factors outside of it (review de Rooij 2015). Recently, an important role
in the regulation of SSC maintenance and differentiation has been detected with
respect to peritubular macrophages (DeFalco et al. 2015). Further studies are needed
to understand how macrophages and Sertoli cells orchestrate SSC behavior in- and
outside of the niche. Unfortunately, in view of the lack of data on the nature of pri-
mate SSCs and also the very high density of Ap and Ad spermatogonia, the nature of
the primate SSC niche remains unknown.
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Another random document with
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he, too, intended for an innocent dupe. The fate of every rich and
lonely woman!
It was under the Christmas tree at Lakemere that Harold Vreeland
learned for the first time why the Queen of the Street had held him
for months in a glittering quiescence in the rapidly built-up firm.
The merry guests were already assembled on the other side of the
curtain when the breathless Justine drew Vreeland into a dark
corner.
The French woman’s panting bosom heaved as she whispered: “She
wants to see you in there, first of all. Now is your time, but don’t
forget me, Harold.”
There was the pledge of an infamous pact in the meeting of their
guilty eyes. Justine now stood, with flaming sword, between her
secret lover and those who would approach the woman who held
both their fortunes. Her dark fidelity was doubly bought.
It was a robed queen who stood waiting there by the fragrant
Christmas tree and held both her hands out to Harold Vreeland. The
Lady of Lakemere at her very best!
With beaming eyes, she handed him an envelope and whispered:
“The time has now come when you will have your own part to play,
under my sole orders.
“I know your whole record. You have been my own faithful knight.
“Listen! All these merrymakers will go away with the old year. Judge
Endicott brings me the firm’s settlement papers on New Years. I will
then send for you and make you my secret representative in a
momentous affair.
“To protect my interests you must at once leave the Waldorf.
“Trust to me!” she smiled. “I will have your bachelor apartments
ready. And no one, not even Wyman, must ever learn of your ‘secret
service.’ Silence and obedience, and your fortune is assured. You
alone shall battle for me and drive this fool Hathorn forever from the
Street.
“Go now! You will leave with the first departing guests, but await my
telegram at the Waldorf to come to me here. And so, I have your
plighted word. Never a whisper to a living soul. You are to be still
only the office partner—to the world!”
Vreeland snatched her trembling hand and kissed it.
It was burning in fever.
But he sped away, and before the curtain rose to a chorus of happy
laughter and shouts of delighted surprise, a glance in a corner of the
hallway where Justine awaited him showed him a check for twenty-
five thousand dollars as his Christmas gift. His patroness had
handed him the precious envelope in silence.
In a low whisper he opened the gates of paradise to the French
woman, who watched her lover with flaming eyes. “Five thousand
dollars of this to you, if you find out for me the secret of that child.”
And he left her, panting with the thrill of a sated avarice.
“I will go through fire and water to serve you,” she faltered. “I will
steal the secret from her midnight dreams.”
That night, after the gay dances were done, when the house was
stilled, Elaine Willoughby sat before her fire, while Justine laid away
the regal robes.
There was the glitter of diamonds and the shimmer of pearls
everywhere. With her hands clasped, the lonely mistress of
Lakemere gazed into the dancing flames.
“I must crush him—to leave the past buried—that I may yet find the
path trodden by those little wandering feet.
“Ah, my God!” she moaned, “it is not revenge that I want. It is love—
her love—I burn to know her mine alone. And the past shall be kept
as a sealed book, for her dear sake. It must be so. It is the only way.
For, Vreeland is brave and true!”
The handsome hypocrite was even then dreaming of a “double
event,” a duplicated prize, one beyond his wildest hopes.
“By heaven! I’ll have both her and the fortune!” His busy, familiar
devil “laughed by his side.”
BOOK II—With the Current.
CHAPTER VI.
IN THE “ELMLEAF” BACHELOR APARTMENTS.
Mr. Harold Vreeland silently wondered at the protean character of

womanhood as he watched Mrs. Elaine Willoughby’s almost feverish
gaiety in the few days which followed their new relation. “I can
believe anything of a woman’s subtle arts after this,” he wonderingly
said, as he made himself a graceful factor in the joys under holly and
mistletoe.
Not a single reference to the coming business had ever escaped her,
and she was as merrily impartial in her favors as a girl at her first
ball.
There was hardly a financier in the house party, Noel Endicott’s visit
of a day being a mere duty call. “She evidently wishes to hide our
intimacy from all, and to publicly impress only the social character on
our friendship,” mused Vreeland.
“Lady mine! you are a deep one!” he mused. Never had he been
placed next to her at dinner, and even at the cotillion’s trifling favors
she had only sought him among the very last.
He was aware that all New York knew of how Hathorn had been
coached up by her into financial glory. “That mistake she will not
repeat. It’s a case of blood and the secret warpath, now,” reflected
Vreeland.
But with the check for twenty-five thousand dollars in his pocket, he
felt that his silence was paid for in advance. He was swimming gaily
on with the current now.
He had calmed Dr. Hugo Alberg down into an expectant friendship
by agreeing to dine with him once a week and to exchange regular
reports at Martin’s.
“So far, I have found out nothing, Doctor,” said the lying Vreeland;
“but I have written out West. I understand that she has immense
properties in Colorado. If I get a clew, you and I will work it up
together.
“In the meantime watch over her yourself, and then tell me all.”
Alberg was in high glee at his social début, and confided to Vreeland
that their patroness was in the most brilliant health.
“Well,” good-humoredly answered Vreeland, “between you and me
we will manage to guard her and take care of her property, if she will
only let us.”
The miserly German’s eyes gleamed as they drank to their private
pact.
“The great thing is to keep every one else away from her,” whispered
Alberg. “I of course watch her professionally. You must keep off all
those society sharks like that upstart Hathorn.”
“Trust to me. I know my business,” laughed Vreeland.
When Vreeland left Lakemere on the last day of the old year, his only
reminder was the whispered word, “Remember!”
“I will wait at the Waldorf,” he answered, with a last meaning gleam
in his eyes.
Standing now on an almost secure pedestal, Vreeland never dared
to dream of the sentimental approach upon the woman who was now
the sole arbiter of his destiny.
“That would be the clumsiest mistake of all,” he decided. “Only—
when she comes into the net—I will tighten it.”
He well knew that the bachelor apartment was to be the scene of
some veiled financial strategy and not a rosy Paphian bower.
Mean and low at heart as he was, he knew that her soul towered
above all low deceit, as the rosy Jungfrau lifts its unsullied peaks to
the blue skies.
The one rare virtue of this scoundrel in embryo was that no inane
self-conceit clouded his clear and mercilessly direct reasoning.
“I am only her tool as yet,” he reflected.
“But the Endicotts, Aubrey Maitland, and Wyman are to know
nothing! This, of course, excludes Senator Alynton. She trusts me
then alone—of the whole world.” And so, he panted for the coming
solution of the enigma.
As he departed for New York, he left one lynx-eyed aid behind him.
It was the hot-hearted Justine, who already knew what her étrennes
would be—a second thousand dollars.
“Remember, my own Justine,” he urged, with glowing eyes, “I want
you to earn that whole five thousand dollars. Spy on Alberg.
“He is only a fool. But catch every whisper of this wary old Endicott.
He alone knows what you must learn. There lies your fortune—in
those pretty ears.”
“I have made my plan,” smilingly whispered the brown-skinned maid.
“I am going to earn that money. Remember, I am vraie Française!”
There were but two plausible explanations of Madonna’s strangely
secret course. Some “deed without a name” was in the plot.
And none of her old friends, no one of the financial world, not even
her advisers, tried and true, were to share the secret of the two for
whom the “bachelor apartment” was to be the veiled headquarters.
“Either Hathorn has surprised the secret of her early life and spirited
away her child, hoping to have drawn her down to his will, or—can it
be that she means to sell out the sugar syndicate?”
He gazed reflectively at the winter landscape flying by, as he
pondered over the first. “No!” was his correct deduction. “Hathorn is
powerless. He would surely have held on to her business, by fear or
force, and gained the inside track in the great gamble, Sugar.
“He would have bent her to his will, and carried on the old hideous
gilded sin—to make his light o’ love his young wife’s best friend.
“That’s quite fin de siècle in New York, they tell me.
“His crime was only the bull-headed folly of preferring another and a
younger woman to the one woman whom he could have gratefully
and logically married.
“There is a man’s fatuous vanity to look for her support in chasing
down a younger and prettier rival under her very eyes. And yet, she
hastened on the marriage. By Jove! she did. It’s a mystery to me!”
The puzzled schemer never knew of the real foundation of his own
blossoming fortunes.
The lightning flashes of Elaine Willoughby’s mad anger came when
Judge Endicott had found out that Frederick Hathorn was secretly
shadowing his loving and trusting employer and tracing back her
past life. Then, Elaine Willoughby became a wolf on the trail.
All of social life is but a hoodman blind game. The stern old lawyer
was only carrying out a secret quest, and he could not divine that
Hathorn’s real object was to trace down to a direct connection Elaine
Willoughby’s secret alliance with the heads of the great Sugar Trust.
It was but a mean money greed, after all.
Keenly alive to every pointer of the Street, Hathorn had not learned,
for all his five years of florid devotion, that his lovely patroness had
sealed that one treasured secret in her soul. And, meanly, he had
tried to dig under her mines. But it was the thirst for gold alone that
fired his veins.
Hiram Endicott alone knew of the coolly-devised scheme by which
the woman whom he had deceived had made Hathorn’s marriage
and enforced absence the epoch for a complete break-off of all
business relations.
But she, deluded, as even the keenest mortals are, in stocks, love,
or war, only feared for the one darling secret of her heart—that veiled
sorrow which lay behind Hiram Endicott’s useless search of years.
And in the defense of that one pure and unsullied memory, she
would have torn herself from Hathorn’s arms even at the altar. For
there is a love which can tear the face of any man from a true
woman’s heart—the mother love.
Before Harold Vreeland reached New York he had decided upon the
“Sugar speculations” as the secret reason for setting up the
“bachelor apartment.”
It was now a matter of gossip on the Street that the great Standard
Oil Company was reaching its octopus arms out for “safe
investments,” as well as sure new speculative fields.
To use a huge surplus of idle money and an inexhaustible credit, it
was rumored that they proposed to sneak into a quiet mastery of the
American Sugar Refinery Company’s outside stock, and even in time
to gather in all the great Gas monopolies of Manhattan Island.
“That’s my lady’s game!” gleefully cried Vreeland. “She could crush
Hathorn at will. Their business is ruined. The ugly rumors as to his
shabby behavior to her have hurt that firm.
“And Potter, too, is about to draw out. Wolfe has no money to speak
of. ‘Missie VanSittart’ will not pay her husband’s stock losses, not
she.”
Harold Vreeland grinned, for the young woman’s name was even
now lightly bandied by the club jackals, but only sotto voce, in
deference to her social rank.
“That’s her little game! She dares not let Alynton know that she
proposes to dally with the other side in this coming fight of giants.
“She proposes to ‘copper’ the game of ‘more sweetness and light,’
as well as play it for a sure winner.
“And I am to be her left hand, and the right (our firm) is to be ignorant
of what the left doeth!”
He was well aware that in some way she was always early
possessed of all the Sugar Syndicate secrets. Whether Endicott,
Alynton, the statesman, or Conyers was her coach, he knew not.
“Perhaps all three—the law, the senate, and journalism,” he grinned.
“Or, is she going to betray these people to the newcomers? There
would be enormous profits in that ‘sweet surrender.’
“I shall enjoy the run, and be in at the death,” he gaily cried, as he
sought his hotel in a vastly enhanced good humor.
When he entered the Waldorf, a sudden encounter made him think
that angels of light had invaded the crepuscular gloom of the long
hall in the early winter evening.
He scarcely credited his senses when Mrs. Frederick Hathorn
motioned him to a corner of the Turkish room, after his startled but
gravely polite salute.
The lady was a ravishing example of the “survival of the fittest” in the
line of Worth, Pingât, and Redfern.
With a wave of her slender hand she swept away the flood of easy
lies that were trembling on his lips, and then, her bright eyes, deep
and searching, seemed to delve into his very soul.
“Remember, you need not answer, if you don’t care to; but you must
give me the right to feel that you are a gentleman. My husband’s
enemies may or may not be mine!” She burned a Parthian glance
into his trembling heart.
“Why did you conceal from him that you were going into banking,
and let him go on and parade you as his best friend, and all that?”
she curtly demanded.
“Mrs. Hathorn,” earnestly pleaded the accomplished liar, “another
man had my promise of partnership long before I left Montana. He
was already making arrangements as to offices and several
confidential matters on which our firm’s future depended.
“I was only asked to give your husband an answer on October 1st.
“And I did give one early enough—a friendly declination—which your
husband made me repeat too forcibly. You should not blame me for
our little estrangement.”
“And you did not scheme to supplant him with—with—”
“One word, Madame,” meaningly said Vreeland. “I have opened my
heart to you. Noblesse oblige! Your question was one I could
honorably answer, just as I could not honorably forewarn men whom
fate had made our business rivals.
“Remember, all’s fair in love, stocks, and war. And it’s every man for
himself in New York.”
“And every woman, too,” added Mrs. Alida, with a little snaky smile.
“I always liked you,” she hesitatingly said, and then, her eyes
dropped before the ardent and merciless gaze of a man who now
saw “a new way to pay old debts.”
“Then let me volunteer a hint that I have never supplanted your
husband with anyone nor sought to. My capital alone gives me
weight in our firm. I have never even opened a ledger or made an
individual transaction.
“Pride and old comradeship should temper each other in your
husband. I have only avoided the distressing embarrassments which
he alone has brought upon himself, and you will always find me your
secret friend, though I can not seek you out in this witches’ parade of
Gotham. You know all the reasons why I can not range myself
openly at your side.” His devilish familiar was now whispering in his
ear.
The victim was young, fair, and foolish.
The luxurious woman sighed. All her allures had been baffled.
“If I should need to tax your friendship?” she slowly said, her
breathing quick and fierce, telling of her agitation.
It was now Vreeland’s eyes which “burned into her spirit’s core.”
“Yes; if you make the place where we can meet without a foolish risk
and placing us both in a false position.”
He glanced around the debatable ground of the Turkish room. Any
notable, from Chauncey Depew down to the last “Western bonanza
beauty,” might surprise them there at any moment.
“Will you come, if you need me?” he whispered, taking her hand,
with insidious suggestion.
“Perhaps,” she faltered, with pale and trembling lips, and when he
looked up he was alone, but a knot of lilies of the valley from her
corsage lay in his hand.
He dreamed strange dreams that night, for he well knew of the
seraglio secrets of the bachelor apartments of New York.
He knew the light vanity that might lead her feet to the “Castle
Dangerous” to solve the riddle which was as yet a mystery to
himself.
But he whistled “Donna è Mobile” very contentedly, as he awoke to
find a telegram on New Year’s morning from his sly partner in an
already plotted treason. Justine’s words were pithy, and Justine was
on guard.
“Watch in South Fifth Avenue for news!”
The simple signature J followed the words: “Lawyer here. See me
first on your arrival.”
“I think that I am a pretty lucky devil,” was the flattering unction with
which Vreeland regaled his soul, as he drove down to Wall Street
and found the two responsible men there on duty.
Noel Endicott, with Aubrey Maitland, were in a secret junta busily
opening the new set of books.
The finely assumed carelessness of Vreeland covered his desire to
trace and locate the only man he feared—Mr. Horton Wyman.
“Just write your dispatch,” calmly said Endicott. “I’ll send it down to
Washington to Alynton’s private secretary, and you’ll have Wyman’s
answer at once. They are inspecting some of Alynton’s West Virginia
properties.”
And, in truth, an open answer to Vreeland’s holiday greeting was at
the Waldorf when the schemer finished his New Year’s dinner, and
slipped out to visit the lair where a wrinkled hag guarded Justine
Duprez’ convenient pied à terre.
He marveled long over a second dispatch which awaited him there.
Its words were ominous, and yet, Elaine Willoughby was firm and
steadfast in her purpose.
“She has been greatly excited. Doctor Alberg has been telegraphed
for. You are to come to-morrow. Lawyer leaves to-night. Dare not
communicate by letter. I have the most important news.”
And all night Harold Vreeland was tossed about in a vain unrest.
He knew that his greedy accomplice Justine would easily handle and
draw out Alberg, for, not of a jealous disposition, Vreeland was
perfectly willing that the doctor should be made a tool of the facile
Parisienne.
“You know how to lead him on, Justine,” was Vreeland’s easy-going
hint, whereat the demure maid dropped her lashes and smiled. And
after all, even the artful Justine was only means to an end.
The recurring excitement of the tortured lonely rich woman was only
a new phase of the old invincible mystery. “Can it be a gigantic game
of blackmail, the infamous price of someone’s silence—the bribe to
her quiet enjoyment of a station not her own? What hideous
Frankenstein hides behind the dropped arras of her life?”
Vreeland knew how many dark shadows lurked behind the bright
curtains of New York’s Belgravia and Mayfair.
“By God! I shall know soon!” he growled, as the second day of the
new year brought him the dispatch confirming Justine’s watchful
foresight. There was no uncertain ring to the words of the Lady of
Lakemere.
“Come to me at once! Immediate action necessary!”
“It seems to be something recurrent—something, too, that even
golden-massed money can not help! She would quickly brush this
trouble away, if the ‘long green’ would do it. And, it must be
something which that old fox, Endicott, can not help, or he would
stay up there on guard and help it, instead of leaving her to Dr. Hugo
Alberg’s chloral, chlorodyne, and phenacetine. There must be an
ugly snarl—some olden shame, some hidden disgrace.”
Vreeland very well knew that the sly German practitioner’s drugs had
brought his patient “no surcease of sorrow,” but only “pushed the
clouds along” to a new day of reactionary misery. For, the proud
heart was sealed and yet surcharged almost to its breaking.
“I wonder what our opinion of each other would be if we all had to
walk abroad with our life stories openly branded on our faces?”
mused the anxious Vreeland, as he drove away to the station. The
flying wheels of the coupé seemed to clatter “Not much! Not much!
Nothing at all!” He meanly believed all men and women to be as
base at heart as himself.
“Thank Fortune for the decent lies of a smooth appearance and a
still tongue!” was the schemer’s cheering conclusion, as he finally
dismissed all vain moralizing, and then, wondered how he would
meet the crafty Justine first, and so be able to gain her budget of
stolen tidings before he faced the watchful Lady of Lakemere.
The provokingly suggestive face of the French woman met him at
the front door, on his arrival. “I am to see that you have your
breakfast,” she smilingly said, “while Doctor Alberg, now upstairs,
prepares Madame for your visit!”
And, as she drew him into a coign of vantage, she whispered, “He is
now my very slave! For he, too, is hunting for an ally, and I had him
all to myself last evening! Now for the news! You can afford to be
liberal to me!”
Her eyes were beaming with that vicious triumph of an unfaithful
underling discovering the naked soul of her helpless mistress.
The household traitor is the lowest of all human spawn.
“Tell me everything,” was Vreeland’s hurried response. “When I
marry her, I will be the King of the Street, and you shall stay with us
as long as you wish—it will be always the same between us.”
The woman’s gleaming eyes softened in a glow of triumph.
“And, your house at Paris shall be my pied à terre. But, give me
every word—remember!”
His bosom was rent with thronging hopes and fears.
Justine smiled, as she pressed herself close to the handsome
scoundrel.
“It is a bargain, then. I will hold you to every word, and I will never let
you go. How I secreted myself so as to hear them, is my own
business. But I did. In their fear of Doctor Alberg, whom Endicott
heartily despises, they fell easily into my hands. There was much
talk of the child—a girl—a missing girl—her child—but no time or age
was given—no whisper of the father’s name.”
Justine paused, while Vreeland hoarsely whispered, “Go on! Go on!
Quick! She may send any moment for me, now!”
“It appears she had placed her child, to get rid of it, in a ‘private
Orphan Asylum’ here, in the city. Just why—I can not tell. But, they
have lost all traces of the girl.
“The old man said, ‘I have followed up every “private inquiry”
possible, since your orders of last summer. These detective fellows
all come to me for their money on the New Year. Now you must be
brave, Elaine,’ he said, and I could hear her choking sobs.
“‘I have very bad news.’
“‘She is dead?’ almost screamed Madame.
“From my little peep-hole, I could see the old lawyer take her hands.
“‘We have been led away on a blind trail. And I have only just now
found the right one—but to lose it forever, I fear. For one of my best
agents has discovered that Doctor McLloyd’s private Orphan Asylum
was chased out of New York City by some angry rivals, and a bevy
of too inquisitive reporters.
“‘The Doctor was making money too fast, and there was a very
suspicious mortality among the little girls. It reappeared as the
“House of the Good Samaritan,” at a little village in Westchester
County. The old Doctor has gone either to his reward, or
punishment. Probably, the latter.
“‘But the sly hypocrite who sported a D. D., as well as a medical title,
left the Westchester property to a buxom grass widow, who long
officiated as his matron.
“‘This woman is now forty years old—well-to-do—and is married to a
thrifty young farmer—a man who is not particular as to how she
earned the handsome property which they enjoy.’
“Mrs. Willoughby was softly sobbing when he finished.
“‘By the lavish use of money and a compact of immunity, she agreed
to privately examine the books and records. Of course she has not
got them. “It is a friend—and so on.” We did not dare to force her.
For, she is a very sly bird.
“‘She was only twenty-two when she eased the good Doctor’s lonely
hours, and she is a remarkably cool hand at the game of life.’
“The old man sighed, as he said, ‘It appears that the enthusiastic
childless woman, who first adopted the child, died in two years after
your babe was taken away from the “private Orphan Asylum,” and,
as her young husband remarried in a year, he brought the pretty
child back and left her again on McLloyd’s hands—with a handsome
present.
“‘The girl was a beautiful three-year-old fairy, and the “matron”
remembers her especially. But, McLloyd, always anxious for profit,
gave her to the first decent applicants, a respectable childless
couple, from Western New York.
“‘Under the name of “Alva Whiting”—full orphan—she was sent away
for the second time.
“‘I must find her! You must advertise!’ cried Madame.
“‘Ah!’ said the old lawyer. ‘There is no hope. The young widower
came back, after his second wedding was all safe, returning from a
three years’ visit in Europe. By a strange chance, his later union was
also childless.
“‘Rich, and now thoroughly independent, he, too, wished to trace
“Alva Whiting,” and to reclaim her.
“‘But the matron says that even the wily McLloyd, tempted by a
handsome reward, failed to find her. The Western New York people
may have given a false name, to prevent the child from ever knowing
that she was not their own.
“‘That is all—the matron would gladly earn our offered money—she
knows nothing.
“‘And now, God alone can, in his infinite mercy, ever reclaim “Alva
Whiting.”
“‘I have tried to induce this woman to meet me. She flatly told my
agent that she would have all the books burned—and then, deny
everything—if her prosperous middle age was connected with old
McLloyd’s baby farm. She cried, “I’m as fond of money as anyone—
but there’s the end of it.”
“‘No more would she say. I fear there is no hope.’ The old lawyer
was almost in tears, as he saw Madame’s sufferings.
“Now,” whispered Justine, “I merely saved my place by that agility of
body which you have so often praised. When Madame fell in a dead
faint, and the old lawyer screamed for help, I just ran around the hall,
knocking over a table, and—the rest has been left in Doctor Alberg’s
hands. So, you now know all.” The schemer’s brain was working like
lightning as they parted in silence.
After the breakfast, it was Justine who conducted Vreeland to
Madame Willoughby’s morning room—but not until Doctor Alberg
had first waylaid him.
“The same old mystery!” the German sighed.
“Money trouble it can not be.”
He flourished his arm in the direction of the fortune in art and
bibelots scattered around.
“Her general system is without disease. Her mind and self-control
are perfect. She has no concealed bad habits, like the ‘suffering New
York dames,’” he sneered. “But always periodical violent storms of
sorrow, these violent attacks following old Endicott’s business visits.
“There must be some old bug-a-boo. Now, I know lonely women are
often apt to be hysterical.
“Marry her. Take her away to Europe. Make her happy. She is a
Venus de Melos, yet in her prime. Break off the domination of this old
legal crab.
“Marriage reveals all secrets, finally. You will surely break his hold on
her. You are no sentimentalist. I will prescribe marriage—do you
see? We will work together. I will be your ally—your slave—your
friend.
“You can well afford to be generous, and you then can work me in,
as confidential physician, into that golden New York circle where the
women’s confidence once gained, a man need not look further for a
Golconda.
“Discretion, silence, and a willingness to go through fire to hide any
woman’s secrets—voilà—the perfect doctor—the successful medical
man! Shall we work together—you and I?”
The young schemer grasped the greedy German’s hand. “I’ll stop at
nothing—to help you. By God! you shall have her every secret.”
“It’s a go. Loyal to the end,” whispered Vreeland.
“And you and I breakfast at Martin’s every Sunday till I am a happy
bridegroom.”
The alert physician glided away to where Justine Duprez’s eyes
called him, with their velvety lure.
Harold Vreeland’s face was lit up with a tender sympathy, as he knelt
before the fair woman who lay in a chaise longue before her
superbly sculptured fireplace. There was a surprise in store for “the
knight of the ribbon blue.” His patroness’ face was stoically calm.
She was no hysterical weakling!
With a perfect self-possession, she plunged at once into the
“business in hand.”
“A modern mystery,” he murmured. “A sphinx of the heart,” for Elaine
Willoughby, Napoleon-like, ignored her ailments, whether of a mere
passing weakness or “memory’s rooted sorrow.” And at what secret
cost?
“Time presses!” she said. “Follow my every word. For, I have sent
them all away. There is only Doctor Alberg in the house—and Justine
always keeps him in her eye.”
Vreeland breathed hard. It dawned upon him that the universally
pliant French woman was in the receipt of several salaries.
“She is a smart devil,” he thought, with a glow of pride.
“I shall have to travel some, this spring—in connection with very
important movements and reactions of a specialty in the stock
market, which you alone will know of.
“You and I must be the only ones to handle the concealed intimacy
of ours. Not even Endicott suspects.
“He never must. No one in the firm. Should foul play or awkwardness
reveal it, the firm name of Wyman & Vreeland would come down in a
day. So, mark my every word. I will not return to New York till you are
installed in your new apartments in the Elmleaf.
“It is well located, in the ‘thirties’—I have taken the best one on the
top floor to aid you in the gay and showy life that I wish you to lead
there.
“You will find your new man, Bagley, a veteran London valet, just
brought over, waiting to report to you now at the Waldorf. He has
never been in America before, and none of our opponents know him.
I had Justine engage him through Low’s Exchange.
“The rooms are already decorated and arranged for you. They have
been ready for a month, and no one can ever trace the ownership.
The whole belongings are yours.”
Vreeland found a voice. He began his grateful praise.
She smiled faintly. “Nay! no thanks! It is purely a business matter. If
you wish anything else, give a list to me. I will transmit it to the
furnisher, who will at once provide. There will be no bills. I hope that
the rooms will please you. Of course, I shall never see them.”
The young would-be bridegroom noted the cold dignity of her
measured sentences. There was the thin icy rivulet still between
them.
“Mark me now,” she said. “There is a working room, and a private
secretary (a stenographer, typewriter, and telegraph operator) will
attend daily, from nine until six. The telegraph and private telephone
wire are connected with my rooms in the ‘Circassia.’ There is a
duplicate ’phone joining you with ‘Central,’ for your social use.”
Vreeland began to take his cue.
“From the café in the Elmleaf, you can have morning service, and be
furnished such entertainment as you may wish to give. I presume
that you dine at your clubs, or in society. The café accounts will all
come to me.”
She paused, and studied Vreeland’s face closely.
“And, in all this, what am I to do?” he asked bewildered.
The Lady of Lakemere laughed gaily at his embarrassment.
“You are to move in there, at once. Sign the lease for a year. Bagley
will take charge of you. There you are to give a jolly house-warming.
Call up all your friends. Do not neglect Merriman, Wiltshire, and
Rutherstone. You are to be pretty gay. These gentlemen may even
bring some of the ladies who wear diamond garter buckles. So much
the better!
“When I return to New York, you are to report to me at the
‘Circassia,’ and the serious business of your new life will then begin.
The woman whom I will send to you as secretary is thoroughly
reliable, and I will answer for her fidelity.”
“The woman?” babbled the astounded Vreeland.
“Yes! Miss Mary Kelly—a talented young business woman of perfect
accomplishment, who represents me.” The voice of the lady was

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Library of Congress Control Number: 2017958850

© Springer Science+Business Media LLC 2017

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Spermatogenesis is a complex and highly efficient process producing millions of

Pullman, WA, USA Jon M. Oatley

Part I Spermatogenesis in Mammals

Part II Postnatal Development of the Spermatogonial Population

3 Setting the Stage: The First Round of Spermatogenesis���������������������� 39

Part III Spermatogonial Stem Cells

Part IV Spermatogonial Differentiation

Part V Genome Integrity of Spermatogonia

Part VI Tools to Study Spermatogonial Biology

Part VII Therapeutic Potentials and Applications of Spermatogonia

Norman Arnheim Molecular and Computational Biology Program, University of

Cathryn A. Hogarth School of Molecular Biosciences, Washington State

D.G. de Rooij, Ph.D. (*)

© Springer Science+Business Media LLC 2017 3

The present knowledge on primate SSCs and spermatogonial multiplication is

1.2 The Organization of the Seminiferous Epithelium

As described above, spermatogenesis encompasses a great many subsequent types

1.2.1 The Clonal Organization of the Seminiferous Epithelium

1.2.2 Timing of Germ Cell Development

1.2.3 Stages of the Cycle of the Seminiferous Epithelium

immunohistochemical staining for acrosin in the acrosomes, which gives a rather

1.2.4 Wave of the Seminiferous Epithelium

1.3 Spermatogonial Cell Types

1.3.1 Non-primate Mammals

a rarefaction zone in the nuclei. Unfortunately, the distinction between Ap and Ad

1.4 Cell Kinetics

Comparing spermatogonial multiplication in primates and non-primate mammals,

1.5 Regulation of the Epithelial Cycle

As described above, the proliferative activity of the As,pr,al spermatogonia strongly

1.6 The Spermatogonial Stem Cell Niche

spermatogonia probably have another type of niche in a different location as they

Clermont Y (1966) Renewal of spermatogonia in man. Am J Anat 118(2):509–524. https://doi.

IN THE “ELMLEAF” BACHELOR APARTMENTS.

Mr. Harold Vreeland silently wondered at the protean character of

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Pullman, WA, USA Jon M. Oatley

3 Setting the Stage: The First Round of Spermatogenesis�� 39

1.2 The Organization of the Seminiferous Epithelium

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1.2.2 Timing of Germ Cell Development

1.2.3 Stages of the Cycle of the Seminiferous Epithelium

1.2.4 Wave of the Seminiferous Epithelium

1.3 Spermatogonial Cell Types

1.3.1 Non-primate Mammals

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