Spermatogenic CellsdStructure

Jacques Auger, Université Paris Descartes, Paris, France

© 2018 Elsevier Inc. All rights reserved.

Introduction

The male reproductive capacity of adult mammals depends on the ability of their testes to produce viable spermatozoa, as well as,
an adequate level of several hormones including testosterone necessary for the development of libido and reproductive functions.
The formation of the male gamete within the seminiferous tubules is a highly complex process, in preparation from embryonic life
(migration of primordial germ cells), fetal, neonatal and prepubertal (production of gonocytes, prespermatogonia) which develops
from puberty and continues throughout life through the renewal and division of spermatogonia stem cells. Differentiating germ
cells are distributed in a highly organized manner as they move between adjacent Sertoli cells, from the basal to the adluminal
compartments of the seminiferous tubules following the successive developmental stages: (i) A-spermatogonia,(ii)
B-spermatogonia, (iii) primary spermatocyte, (iv) secondary spermatocyte, (v) spermatid and, (vi) spermatozoon (Fig. 1).
The general organization of spermatogenesis is essentially identical in all mammals and can be divided into three main phases,
each of which leads to the production of a distinct category of germ cells:
(1) the mitotic phase which corresponds to the proliferation of spermatogonia. During this phase, spermatogonia produce two
types of diploid cells: stem cells, necessary for the renewal of their own stock, and spermatogonia that proliferate in cascade to
generate differentiated spermatogonia. These latter generate in their final phase primary (preleptotene) spermatocytes,
(2) the meiotic phase characterized by two successive cell divisions without intermediate DNA synthesis and which results in the
formation of haploid spermatids. In all species, this phase is divided into five successive stages called leptotene, zygotene, pachy-
tene, diplotene and diacinese,
(3) the spermiogenetic phase which corresponds to the metamorphosis of each spermatid into a spermatozoon.
Table 1 summarizes the main cellular types and morphological transformations along the spermatogenetic process. At the end of
these three phases, the spermatozoa are released into the lumen of the seminiferous tubules, a process called spermiation. The
morphology features of germ cells and somatic cells of the seminiferous epithelium, tissular organization and synchronic distribu-
tion along spermatogenesis have been evaluated over the years in many species (see notably, Clermont, 1963; De Kretser and Kerr,
1988; Russell and Ettlin, 1990). The main studies involving human spermatogenesis were performed around the 60s by Clermont
and colleagues, who described GCs, spermatogonial kinetics and cell renewal. This article will concentrate on the morphological
and structural features of the male germ cells in mammals with special reference to humans.

Fig. 1 Germ cell organization within a seminiferous tubule (transverse section) with corresponding spermatogenetic phases.

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54 Testis Cell Biology j Spermatogenic CellsdStructure

Table 1 Summary of the mammalian germ cell types along the spermatogenetic process

Mitotic phase
A proportion of spermatogonia enter mitosis and, after five mitosis, become type B spermatogonia, which divide into primary spermatocytes
A1 Spermatogonia/ A2 Spermatogonia/ A3 Spermatogonia/ A4 Spermatogonia
/(Intermediate spermatogonia)/ B Spermatogonia/ Primary spermatocytes
Meiotic phase
Primary spermatocytes enter meiosis. The first meiotic division leads to secondary spermatocytes and the second division to the haploid round
spermatids
Primary spermatocytes (2n chr/4n DNA)/ Secondary spermatocytes (2n chr/2n DNA) / Round spermatids (1n chr/1n DNA)
Spermiogenesis
The spermatogenetic step where spermatids differentiate into spermatozoa:
• DNA is associated with the protamines nuclear basic proteins and become highly condensed
• The acrosome, tail and the midpiece containing mitochondria are formed
• Most of the cytoplasm is eliminated
Round spermatids / Elongating spermatids / Elongated spermatids / Spermatozoa

Spermatogonia

In all mammals, spermatogonia are defined as constituting the mitotic compartment of spermatogenesis including stem, undiffer-
entiated and differentiating cell types, possessing distinct morphological and molecular characteristics. Even though the real nature
of the spermatogonial stem cell and its regulation is still debated the general consensus holds that in steady-state spermatogenesis
the stem cell compartment needs to balance differentiation versus self-renewal.
Spermatogonia derived from the differentiation of gonocytes. In adult mammals, spermatogonia are very few in number and
located in the periphery of the seminiferous tubules, leaning against the lamina propria, in contact with the Sertoli cells. In primates,
such as monkey and man, there are three morphologically distinct subpopulations of spermatogonia: dark type A (Ad spermato-
gonia), pale type A (Ap spermatogonia) and type B (B spermatogonia). In humans, Ad spermatogonia represent the reserve of stem
cell, and Ap spermatogonia correspond to a population of cells intended to ensure the renewal of the stock of the progenitor cells of
the spermatogenetic process. The Ap and Ad spermatogonia are considered to be the counterpart of the mouse undifferentiated sper-
matogonia and type B that of the mouse differentiating spermatogonia (Fig. 2). Ad spermatogonia does not divide under normal
conditions of testicular function. On the other hand, Ap spermatogonia divide by mitosis to maintain their own cellular stock and
for each of them, generate 2 B spermatogonia, which themselves divide by mitosis and enter into meiosis. In humans, only one
generation of B spermatogonia has been characterized, whereas there are four in the macaque.
Indeed, if the general pattern of spermatogonial organization and their differentiation (from stem cell to differentiated sper-
matogonia) is globally identical in mammals, the spermatogonial system presents many differences from one species to another
in primates, and between primates and rodents (Ehmcke and Schlatt, 2006; Boitani et al., 2016; see Fig. 2).

Fig. 2 A schematic diagram of different cell subtypes within the spermatogonial compartment in the mouse, monkey and man.
 Testis Cell Biology j Spermatogenic CellsdStructure 55

The ability to distinguish different classes of spermatogonia is critically dependent on the morphological characteristics of the
nuclei as revealed on stained preparations of transverse sections of seminiferous tubules by conventional light microscopy.
In humans, from a morphological point of view:
 The Ad nuclei contain a deeply stained dust-like chromatin with a characteristic large central vacuole-like cavity, which, at the
electron microscopic level, appears as a chromatin rarefaction zone; close to the nuclear membrane one or more nucleoli are
found.
 In contrast, the ovoid Ap nuclei contain a fine, homogenous and weakly stained chromatin, one or two nucleoli lying close to the
nuclear membrane and no vacuoles.
 B spermatogonia exhibit the features described for other species, though the human cells are somewhat smaller. Type B sper-
matogonia are characterized by large clumps of intensely stained condensed chromatin under the nuclear membrane of an ovoid
nucleus contrasting with a fine granulated chromatin in the rest of the nucleus.
Spermatogonia as observed by conventional light microscopy have a poorly stained cytoplasm. Studies of whole-mounts of semi-
niferous tubules have demonstrated that they remain connected by intercellular bridges such that large numbers are effectively
linked together. Using the PAS reaction, glycogen is found in Ad spermatogonia.
At the electron microscope level (observations by transmission electron microscopy, TEM), the basal position of spermatogonia
within the epithelium and their extensive contact with the basal membrane of the tubule are clearly evident. The extent of this
contact decreases in B spermatogonia which will eventually lose all contacts with the basal membrane to become preleptotene
primary spermatocytes (see below). All types of spermatogonia are characterized by a relatively electron-lucent cytoplasm and
a paucity of cytoplasmic organelles. Because of incomplete cytokinesis during mitosis, spermatogonia remain connected by inter-
cellular bridges, 2–3 mm in width usually not containing organelles or microtubules.
Attempts have been made to classify spermatogonia at the TEM level, notably in humans. Most investigators agree that it is
possible in humans to identify the type A dark, type A pale and type B spermatogonia, though intermediate forms may occur.
The classification is based on: (i) the nuclear and nucleolar features, (ii) the presence of aggregations of mitochondria, (iii) the pres-
ence or absence of glycogen granules, and, (iv) the presence of crystalloids (in human spermatogonia).
 Ad spermatogonia: oval shaped nucleus with electron translucent region compatible with the nuclear vacuole observed by light
microscopy, small nuclei adjacent to the nuclear membrane, mitochondria lying close to the nucleus with a cristae generally
extending transversally across the matrix, abundant rough endoplasmic reticulum, Golgi complex poorly developed, presence of
glycogen granules, often aggregated, presence of crystalloids (¼ collections of fibrils and tubules aligned along the long axis of
the structure up to  3 mm in length).
 Ap spermatogonia: less contact with the basal membrane, finely granulated homogeneous chromatin, nuclei without vacuoles,
amorphous peripherally placed nucleoli, paucity of organelles, few mitochondria generally isolated or by pairs, glycogen
granules rarely observed, presence of crystalloids.
 B spermatogonia: the spermatogonia having the least contact with the basal membrane, peripherally placed chromatin less
prominent than by light microscopy (due to the thin sections), nucleolus centrally placed, mitochondria scattered without the
intermitochondrial material observed in type A spermatogonia.
In both nonhuman and human primates, some authors suggest the existence of so-called A transition spermatogonia, having no
unequivocal morphological characteristics of either Ad or Ap spermatogonia. In the rat, Type A isolated (Ais) is believed to be
the stem cell, whereas in humans, it is unclear which Type A spermatogonia is the stem cell.

Spermatocytes

The cells in the spermatogenetic process that are involved in meiosis are the primary and secondary spermatocytes (Fig. 3). Sper-
matocytes are the cells originating from the last spermatogonial division (B spermatogonia) from which spermatids, then, sperma-
tozoa with half the chromosome complement of the original progenitor cell will be generated following the meiotic phase of
spermatogenesis. The process of meiosis involves two cell divisions, the first involves primary spermatocytes generating after the
diakinesis secondary spermatocytes which in turn are involved in the second meiotic division producing the haploid spermatids.
Because of the long duration of the prophase of the first division (for example, 24 days in humans), primary spermatocytes are
found on tubules sections, in every stage of spermatogenesis (and, for a given stage different types of spermatocytes may be
observed). By contrast, because the second meiotic division progresses rapidly (lasting about 5 h in humans), secondary spermato-
cytes are rarely observed on tubule sections.

Primary Spermatocytes
The primary spermatocytes arising from B spermatogonia lose contact with the basal membrane of the seminiferous tubule and
migrate in direction of the adluminal compartment by transiently breaking the tight junction complexes of the Sertoli cells. The
primary spermatocytes exhibit the features of the first meiotic division in terms of nuclear morphology. As the spermatocytes
pass through meiotic prophase they become larger and the appearance of the nuclear chromatin alters, reflecting the condensation
56 Testis Cell Biology j Spermatogenic CellsdStructure

Fig. 3 Summary of the meiotic stages with the corresponding cellular types and subtypes.

and movement of the chromosomes as they prepare for meiotic division. The meiosis phase involves several types of spermatocytes
ranging in size (from small, for example, preleptotene, to very large, for example, late pachytene) and presenting typical morpho-
logical features for each of the five stages of the prophase of the first meiotic division, preleptotene, leptotene, zygotene, pachytene,
and diplotene which are clearly distinguished under the optical microscope. Peripherally located nucleoli are frequently seen in
primary spermatocytes. TEM reveals they consist of extremely electron dense aggregations of granules. The cytoplasm of primary
spermatocytes is more electron-dense than that of spermatogonia and contains evenly scattered polysomes and ribosomes. Features
of rough and smooth endoplasmic reticulum are rare. However, during the progression of the prophase, the Golgi complex close to
the nucleus gradually enlarges. Thanks to its high resolution power, TEM has revealed the existence of a subcellular structure, the
synaptonemal complex in the nuclei of primary spermatocytes form a number of species. The synaptonemal complex consists of
two lateral elements that appears as electronic dense fibrils equidistant from a central element consisting of a delicate linear region
of increased electron density (its appearance and evolution is presented below with the different cellular and subcellular morpho-
logical features at each stage of the prophase). The number of synaptonemal complexes is equal to the number of divalents. Though
the synaptonemal complex provides the framework necessary for synapsis, it is thought that the recombination modules are the
vital components in the crossing-over of genetic material.
Primary spermatocytes are joined to each other by intercellular bridges similar to those found between spermatogonia. They are
separated from adjacent Sertoli cells by distinct intercellular spaces that are modified in some regions by desmosome-like structures.

Preleptotene stage
They present a spherical nucleus with an aspect similar to those of B spermatogonia, though smaller. Notably, they lack any chro-
mosomal elements. They are termed preleptotene spermatocytes in reference to the S phase, the preleptotene phase of DNA
synthesis, the process by which each chromosome when it condenses is composed of a pair of chromatids, the total of DNA content
representing twice the diploid content.

Leptotene stage
During this stage, the chromosomes become apparent as single fine threads. TEM has revealed they are attached at each extremity to
the nuclear membrane (giving the “bouquet” aspect) by “attachment plaques” which in fact are the lateral element of the synapto-
nemal complexes assembling and appearing at this stage. Though at this stage the chromatid pairs are present, they are not visible
and will not do so until later in prophase. At this stage, the X and Y chromosomes appear associated with the dense body of the
sexual vesicle, a special condensation of chromatin fibrils.
Ovoid mitochondria aggregated in groups of two or three, with an electron dense material similar to that seen in spermatogonia
are observed at this stage (and at the subsequent zygotene stage).
 Testis Cell Biology j Spermatogenic CellsdStructure 57

Zygotene stage
This stage is characterized by the thickening of the chromosomal elements that begin the process of pairing termed synapsis. Pairing
of the elements of the synaptonemal complexes begins at this stage.

Pachytene stage
The long pachytene stage begins with the completion of synapsis and is associated with further thickening and shortening of
chromosomes. During this stage, exchanges of chromosome material between maternal and paternal homologous chromosomes
occur by crossing over. At the points of crossing over, bridges (chiasmata) are seen in variable numbers. Depending on the chias-
mata, different aspects of the chromosomes can be observed. Nuclear and cytoplasmic growth of pachytene spermatocytes result in
these cells becoming the larger of all germ cells. Fully formed synaptonemal complexes are observed by TEM at this stage.

Diplotene stage
Desynapsis occurs during this phase. The paired chromosomes partially separate (jointly to the synaptonemal complexes disas-
sembly) but remain joined at their chiasmata. Subsequently, the chromosomes further shorten, then detach from the nuclear
membrane during the diakinetic phase, the stage where the two chromatids of each chromosome can be seen.

Diakinesis to telophase
Diakinesis is rapidly followed by the disappearance of the nuclear membrane, the appearance of the spindle and the alignment of
the homologous chromosome pair with the metaphase plate, followed by the migration of each part of the chromosome pair
toward the opposite poles of the spindle during the anaphase. The telophase completely separates the chromosomes and produces
the secondary spermatocytes.

Secondary Spermatocytes
These are the cells that undertake the second meiotic division which begins after a very short interphase without DNA replication
takes place. The phases are typical of any cell division (pro-, meta- ana and telophase 11) in that one chromatid from each pair
(making up each chromosome) migrates to each daughter nucleus. The secondary spermatocytes have the haploid number of chro-
mosomes, though their DNA content is still diploid so that when they complete meiosis, the resulting spermatids have both an
haploid chromosomal content and DNA content.
Secondary spermatocytes are round cells with a size ranging between the primary spermatocyte and the round spermatid size and
a greater nucleo-cytoplasmic ratio. They are situated close to the lumen of the tubule (see figure) and their nucleus contains an
homogeneous chromatin network throughout which large globular chromatin masses are dispersed. Nucleoli centrally positioned
are often observed. Light and electron microscopy reveal the presence of intercellular bridges between adjacent secondary spermato-
cytes similar with those found for other germ cell categories. TEM reveals scattered features of endoplasmic reticulum arranged
concentrically around the nucleus, a prominent Golgi complex containing vesicles with electron-dense granules and dispersed mito-
chondria with dilated intracristal spaces.

Spermatids

Spermatids are the products of the second meiotic division and lack the ability to divide. Their progressive transformation takes
a unique, complex, and lengthy process (for example, 23 days in humans) of morphological and functional differentiation termed
spermiogenesis which sees the early spermatids with a usual cellular aspect (a round cell with a central nucleus surrounded by
homogeneously distributed organelles) at the time of their formation to be metamorphosed into highly specialized polarized cells:
the spermatozoa. Under physiological conditions, the transmission of the paternal genome to the oocyte at the time of fertilization
requires the development of the acrosomal system, the rearrangement of the nucleus and perinuclear structures, the assembly of the
flagellar structures, the elimination of the vast majority of the cytoplasm concomitant with a reorganization of cell organelles. These
changes will be briefly reviewed using human spermiogenesis as a model. The major morphological features of spermiogenesis are
common to all species but, details in the morphogenetic process and the resulting sperm morphological characteristics vary for each
species since different morphological features may depend on different species-specific genetic determinants. The mammalian
sperm morphogenesis is a continuum sequence of complex morphological changes which has been subdivided for descriptive
purposes into successive steps, the number of which varies according to the species (rat: 19, mouse:16, ram, bull, boar: 15, baboon:
10). In humans, six steps are distinguished according to the shape of the nucleus, the aspect of the chromatin and the general
morphology of the cell (Fig. 4): Sa, Sbl (round/early spermatids), Sb2, Sc (elongating/intermediary spermatids) Sd1, Sd2
(elongated/mature/late spermatids).
The first step corresponds to the small round spermatid produces by the second division of meiosis. The round spermatid,
smaller than the secondary spermatocyte presents a central spherical nucleus with a diffuse chromatin containing one or
more nucleoli lacking a clear fibrillary center (subsequently, in spermatids in elongation, the nucleolus fragments before disap-
pearing completely), a well developed Golgi apparatus, adjacent centrioles, dispersed mitochondria lying peripherally close to
the plasma membrane and microtubules (the future components of the axoneme) visible at the periphery of the cytoplasm.
58 Testis Cell Biology j Spermatogenic CellsdStructure

Fig. 4 Spermiogenesis steps in humans. Modified from Holstein, A. F. (1976). Ultrastructural observations on the differentiation of spermatids in
man. Andrologia 8 (2), 157–165.

In addition the chromatid body, a regular electron-dense mass, can be observed adjacent to the Golgi complex. During the evolu-
tion of the round spermatid, the nucleus and the Golgi vesicle attached to it rotate to a position opposite to that of the developing
flagellum.
Spermatids are located in the adluminal compartment of the seminiferous tubule, in close contact with Sertoli cells. Spermatids
at different steps of differentiation may be observed in the same tubule cross section (see Fig. 1).

Formation of the Acrosome

The evolution of the acrosomal material, as revealed by PAS staining, has been one of the criteria used to identify the steps of the
spermatid differentiation by light microscopy. The acrosome morphogenesis is generally described according to four subsequent
distinct morphological phases which can be easily distinguished by TEM: the Golgi, the cap, the acrosome and the maturation
phases. These four phases exhibit a general pattern but display variations in different species. Soon after the formation of the early
round spermatid, the Golgi apparatus assembles the elements necessary for the formation of the acrosome: several small
secretory-like granules rich in glycoproteins form on the trans face of the Golgi stacks. These proacrosomic granules coalesce
to give a single larger acrosomic vesicle which associate with the nuclear membrane and tightly adheres to it to become an acro-
some. This early acrosome show an electron-dense core and a less dense cortex by TEM. The Golgi apparatus continues to
contribute glycoproteins to the acrosome by means of numerous small carrier vesicles. The lighter peripheral portion of the acro-
some expands at the surface of the nucleus taking the shape of a cape covering at this step  half of the nuclear surface. The tight
adhesion of the acrosome membrane to the nuclear membrane is due to the presence of proteinaceous substance that later
condense to form part of a rigid capsule or perinuclear theca that covers the nucleus of spermatozoa. With the displacement
of most of the cytoplasm toward the caudal pole of the spermatid, the Golgi apparatus separates from the acrosome, regresses
and finally disappears. At later steps of spermiogenesis and as the nucleus takes its species-specific shape, the acrosome also
condenses and conforms to the changing form of the apex of the nucleus shape. The final size and shape of the acrosome varies
from one species to another (for example, small in man and important in the guinea pig for example and paddle-shaped in
human vs. hook-shaped in rodents).

Nuclear Elongation and Chromatin Condensation

During spermiogenesis, the central nuclei of the round/early spermatids elongate and condense their chromatin to become
the main polarized component of the head compartment with an acquired species-specific shape (piriform in man,
 Testis Cell Biology j Spermatogenic CellsdStructure 59

flattened or paddle-shaped in ram, bull, and dog, falciform in rodents). A remodeling of the chromatin from its nucleo-
somal form to a thread-like filamentous form is first observed, then, the chromatin filaments thicken, become coarse and
aggregate into compact masses that coalesce to form a dense chromatin mass (as illustrated in Fig. 4). It is established that
the first morphological transformation is associated with histones modifications and loss, the second being linked to the
presence of TP1 and TP2 transition proteins. Late in spermiogenesis, when the chromatin condensation has occurred and
the definitive shape of the nucleus has been acquired, the transition proteins are replaced by arginine and cysteine-rich
protamines conferring to the nucleus an extreme compaction protecting the genome. Several studies have indicated a specific
arrangement of chromosomes in round and intermediate spermatids which could be involved in the formation of the male
pronucleus.

Assembly of the Flagellar Structures

During the early steps of spermiogenesis, the pair of centrioles migrates toward the nucleus. One out of the two centrioles triggers
the formation of the axoneme consisting of a bundle of microtubules, arranged in a 9 þ 2 pattern (the intracytoplasmic axoneme
is observed precociously from step 2 in humans). As the centrioles move toward the nucleus, they pull with them the plasma
membrane, which then forms a double-wall sleeve around the growing axoneme. In the elongated spermatid, electron-dense
material is deposited around the centrioles and forms a striated collar that characterizes the neck portion of the tail. The sperm
tail is not similar to other flagellum or cilium since it associates a periaxonemal component including two specific cytoskeletal
elements, the outer dense fibers and the fibrous sheath which are deposited along the central axoneme soon after it terminates its
growth. The mitochondria peripherally close to the plasma membrane in round spermatids migrate toward the forming tail as the
manchette appears in elongating spermatids. Following the sliding of the annulus (which derives in part from the chromatoid
body) along the axoneme and the periaxonemal structures, the mitochondria line up in an orderly manner along the dense fiber
between the neck and the annulus. Later during spermiogenesis, the mitochondria condense and dispose themselves side by side
and tip to tip in a spiral manner. This mitochondrial sheath delimitates the midpiece of the length whose length is species-
specific.

Cytoplasm Elimination
The gradual elimination of the cytoplasm from early spermiogenesis until the formation of mature spermatids is an evident obser-
vation in seminiferous tubule sections by light microscopy and TEM. In the most advanced phases of spermiogenesis, nearly 90% of
the spermatid cytoplasm is eliminated. This progressive elimination takes place in two stages: (i) the first is continuous via the tubu-
lobulbar complexes, the cytoskeleton-related membrane structures developing at sites of intercellular attachment in the mammalian
seminiferous epithelium, a component of the sperm release mechanism (at apical junctions between Sertoli cells and spermatids the
structures internalize adhesion junctions and evaginations of the elongating spermatids penetrating inside the Sertoli cells), (ii) the
second occurs at the time of spermiation when the main part of the cytoplasm that remains after the reduction of tubulobulbar
complexes is eliminated in the form of corpuscles called residual bodies, which are captured and phagocytosed by Sertoli cells.
These residual bodies contain most of the spermatid organelles, lipids and polyribosomes, and their phagocytosis probably plays
is known to play an important role in regulating the functioning of the Sertoli cell and in synchronizing the cycle of the seminiferous
epithelium.

Transitory Organelles and Structures

Spermiogenesis is characterized by the presence of transient organelles whose role is unknown or not fully elucidated. Cytoplasmic
microtubules anchored on the nuclear ring posterior to the acrosome constitute the manchette. The manchette displaces part of the
cytoplasm with its organelles behind the caudal part of the nucleus and the nuclear ring also moves caudally. It is visible in the
elongating spermatids and disappears well before the end of spermiogenesis. It consists of microtubules which form a sort of mitotic
half-spindle arranged around the caudal region of the nucleus. The manchette, connected by a filamentary tract to the nuclear
envelope and to the chromatin, could exert an external stress on the nuclear form, sliding gradually toward the base of the nucleus
(Lehti and Sironen, 2016). The spindle-shaped body.
The chromatoid body located in the cytoplasm of round spermatids appears like a cluster of electron dense granules that presum-
ably derives from the nuclear membrane, although its significance remains unknown. It contains actin, long half-life RNA, basic
proteins, ribonucleoprotein particles as well as polysaccharides and cations.

References

Boitani, C., Di Persio, S., Esposito, V., & Vicini, E. (2016). Spermatogonial cells: Mouse, monkey and man comparison review. Seminars in Cell and Developmental Biology, 59,
79–88.
Clermont, Y. (1963). The cycle of the seminiferous epithelium in man. American Journal of Anatomy, 112, 35–51.
De Kretser, D. M., & Kerr, J. B. (1988). The cytology of the testis. In E. Knobil, & J. D. Neill (Eds.), The physiology of reproduction. New York: Raven Press.
60 Testis Cell Biology j Spermatogenic CellsdStructure

Ehmcke, J., & Schlatt, S. (2006). A revised model for spermatogonial expansion in man: Lessons from non-human primates. Reproduction, 132, 673–680.
Lehti, M. S., & Sironen, A. (2016). Formation and function of the manchette and flagellum during spermatogenesis. Review Reproduction, 151, R43–R54.
Russell, L. D., & Ettlin, R. (1990). Histological and histopathological evaluation of the testis. St. Louis: Cache River Press.

Further Reading

Oko, R., & Clermont, Y. (1998). Spermiogenesis. In Encyclopedia of reproduction. San Diego: Academic Press.