Spermatids

Barry R Zirkin, Johns Hopkins University, Baltimore, MD, United States

Erwin Goldberg, Northwestern University, Evanston, IL, United States
© 2018 Elsevier Inc. All rights reserved.

Glossary
Acrosome A membrane bound organelle closely applied to the surface of spermatids and spermatozoa. Formed by the Golgi
apparatus of early spermatids, it is rich in hydrolytic enzymes and important for fertilization.
Axoneme The central portion of the sperm tail composed of a microtubular array with associated structural proteins.
Fibrous sheath The cytoskeletal structure along the principle piece of the tail. Glycolytic enzymes important for sperm
metabolism are tightly bound to the fibrous sheath.
Manchette Appears in elongating spermatids as a sheath extending from the nucleus, surrounding the tail and disappearing
during the late steps of spermiogenesis; involved in protein transport to the base of the axoneme.
Outer dense fibers Major cytoskeletal elements of the tail of elongated spermatids and spermatozoa. In the middle piece there
are nine ODFs, one per axonemal doublet. In the principle piece of the tail only seven ODFs are present.

Introduction

As described in the section “Overview of Spermatogenesis,” building a sperm consists of the formation of spermatogonia from sper-
matogonial stem cells, mitotic divisions of the spermatogonia to form spermatocytes, two meiotic divisions of the spermatocytes
that result in the formation of spermatids, and spermatid differentiation to form spermatozoa. The latter process, spermiogenesis, is
the focus of this article.
The round spermatids that result from meiosis exhibit a remarkable transformation during spermiogenesis to become flagellated
spermatozoa with extremely compact nuclei. During this process, the round spermatids undergo profound remodeling in sequen-
tial “steps” to form elongated spermatids. The steps are distinguished from each other by the specific structural changes that occur in
the forming acrosome and the nucleus. In addition to morphological changes, there are molecular events that lead to the formation
of the sperm acrosome, mid-piece and flagellum. The morphological and molecular changes that occur during spermiogenesis are
critically important in achieving production of functional male gametes.
During the cellular transformation of haploid round spermatids to spermatozoa, major structural changes occur that include
formation of the acrosome from the Golgi apparatus, nuclear elongation, chromatin condensation, axoneme formation triggered
by the centriolar complex, and assembly of sperm-specific cytoskeletal elements including the mitochondrial sheath surrounding
the axoneme. The distinct steps of spermiogenesis identified in man (Oko and Clermont, 1999) are depicted in Fig. 1. Recently,

Fig. 1 Depiction of transition steps from round spermatid to elongated spermatozoan. From Oko, R and Clermont, Y 1999. Spermiogenesis. Ency-
clopedia of Reproduction, Vol. 4 Pro-Z, pp. 602–609. Cambridge, MA: Academic Press.

42 Encyclopedia of Reproduction, 2nd edition, Volume 3 https://doi.org/10.1016/B978-0-12-801238-3.64426-4

 Spermatogenesis j Spermatids 43

high-resolution light microscopy (HRLM) has enabled improved morphological identification of all germ cells (spermatogonia,
spermatocytes, spermatids) in the human testis (Nihi et al., 2017). This approach has led to a highly reliable way to identify the
events of spermatogenesis in man. Precise staging of germ cells is important for predicting causes of aberrant spermatogenesis
and infertility.

Spermiogenesis

Spermatid development, or spermiogenesis, extends over 22.7 days in rats and 21.6 days in humans. Formation of the acrosome is
a key event in spermatid development. The acrosome is a membrane-bound organelle on the surface of the nuclei of spermatids and
spermatozoa. Acrosome formation begins in the early spermatid by coalescence of proacrosomic vesicles to a single large vesicle
which tightly adheres to the nuclear membrane. The acrosomal vesicle is rich in hydrolytic enzymes that are released during the
acrosome reaction, a critical step in fertilization that enables sperm penetration of the zona pellucida of the egg.

DNA Compaction

Morphological remodeling of the spermatid involves nuclear elongation and chromatin condensation, producing the species-
specific form that is characteristic of the sperm head. During these processes, there is reorganization of the haploid genome and
compaction of DNA. Human sperm chromatin undergoes a complex remodeling in which histones are replaced by protamines
during a regulated transition that involves histone modifications (H1t histone is modified or lost) as well as intermediate and
temporary replacement of the histones by sperm-specific transition nucleoproteins TP1 and TP2. Chromatin compaction is
influenced by TP1 and TP2. These transition proteins are then replaced late in spermiogenesis by arginine and cysteine-rich
protamine. Protamine polymorphisms can lead to compaction defects, increased susceptibility to DNA damage, and infertility.
The replacement of most histones by protamine 1 (PRM1) and protamine 2 (PRM2) facilitates a high order of chromatin pack-
aging necessary for normal sperm function, and may also be required for DNA silencing and imprinting changes (Carrell et al.,
2007).
Epigenetic modifications, characterized by DNA methylation, histone substitutions, and chromatin remodeling, also are impor-
tant regulators in spermiogenesis. Several genes in the germinal epithelium are regulated through epigenetic mechanisms, and there
have been suggestions of an influence of epigenetics on spermatogenesis. Epimutations (often hypermethylation) in several genes
(e.g., MTHFR, PAX8, NTF3, SFN, HRAS, JHM2DA, IGF2, H19, RASGRF1, GTL2, PLAG1, D1RAS3, MEST, KCNQ1, LIT1, SNRPN)
have been reported to be associated with poor semen parameters or male infertility. Recent reports suggest that epigenetic modi-
fications in mature sperm may also have effects on embryo development (Carrell and Hammoud, 2010).

The Manchette (Acrosome and Tail Formation)

Spermiogenesis also involves the elongation of spermatids and removal of excess cytoplasm, as well as the formation of the acro-
some and tail. Protein transport during spermatid elongation is required for the formation of the sperm tail and acrosome, and for
shaping of the sperm head. Proteins are transported to the developing sperm head and tail by the manchette, a microtubular struc-
ture that surrounds the elongating spermatid head and that is present only during spermatid elongation, and by the axoneme,
a microtubule-based cytoskeleton composed of nine pairs of microtubules doublets and two central microtubules that is the
core structure of the sperm tail depicted in Fig. 2 from Chen et al. (2016).
In the mouse and human, the manchette is first seen first in mid-spermiogenesis (step 8 in the mouse, and step 5 in the human;
see Fig. 1). It forms between the perinuclear ring that surrounds the nucleus and the elongating sperm axoneme. Much is now
known about the assembly, disassembly and function of the manchette, and the roles of these processes in spermatid head-
shaping and sperm tail formation, including the molecular events that are involved (Lehti and Sironen, 2016). Among the molec-
ular players is SPEM1, a protein expressed only in developing spermatids (Bao et al., 2011). Inactivation of Spem1 in mice has been
shown to result in deformed spermatozoa. UBQLN1 has been identified as a SPEM1-interacting partner (Bao et al., 2010). UBQLN1
and SPEM1 have been co-localized to the manchette of elongating spermatids. UBQLN1 functions through binding and directing
poly-ubiquitinated proteins to the proteasome for degradation. Consequently, the interactions shown to occur between UBQLN1
and SPEM1 suggest a role for UBQLN1 in regulating protein ubiquitination during spermiogenesis. Ran-binding protein 17
(RANBP17) also was identified as an interacting partner of SPEM1 (Bao et al., 2011). RANBP17 was shown to localize to the XY
body of primary spermatocytes, later in spermatogenesis to the nuclei of round spermatids (steps 1–7), and finally, together
with SPEM1, to the manchette of elongating spermatids (steps 8–14). As a member of a protein family involved in nucleocytoplas-
mic transport, RANBP17 has been suggested as possibly playing a role in sex chromosome inactivation during meiosis and/or in
intramanchette transport during spermiogenesis. Interactions between RANBP17 and SPEM1 suggest that SPEM1 might function
in RANBP17-mediated nucleocytoplasmic transport.
Observations by electron microscopy have revealed rod-like elements linking the manchette microtubules to the nuclear
membrane (Russell et al., 1994). The manchette appears to be connected through a protein complex called the LNC complex (linker
44 Spermatogenesis j Spermatids

Fig. 2 Formation of Manchette during spermiogenesis. From Chen, S.R. et al. 2016. Cell Death & Disease 7(11), e2472.

of nucleoskeleton and cytoskeleton). Two LINC complexes have been identified during spermatid elongation: the SUN3/Nesprin 1
protein complex, which has been shown to connect the manchette to the nucleus; and SUN1/Nesprin 3 which, with SUN3/Nesprin,
has been localized to sites at which manchette microtubules contact the nuclear envelope. It has been suggested that the LINC
complex may be involved in nuclear shaping (Göb et al., 2010; Calvi et al., 2015).
Successful sperm production requires proteins and protein complexes to be transported to the correct assembly sites during
sperm tail formation. However, the exact roles of microtubule and actin-based delivery complexes remain uncertain. Some proteins
appear to be transported through the manchette to the base of the developing sperm tail. However, not all sperm-tail proteins have
been localized to the manchette, raising the prospect that different, more complex, mechanisms for protein storage and transport
may be involved in sperm formation (Lehti and Sironen, 2016). Functional studies will provide a more complete understanding of
the mechanisms involved in constructing a fertile sperm, important for developing therapies for male infertility and perhaps for
identifying targets for a reversible male contraceptive technology.

Formation of the Tail

Sperm tail formation is an important step in remodeling the spermatid. Early in spermiogenesis, a pair of centrioles migrates toward
the nucleus and eventually binds to it (Fig. 1). Formation of the central component, the axoneme, is initiated by one of the pair. The
axoneme is comprised of a characteristic “9 þ 2” microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regu-
latory complex (N-DRC) (Inaba, 2011). As noted above, there is an intimate association of the nucleus and manchette for delivery
of essential proteins. It currently is hypothesized that proteins are moved by intramanchette transport to the base of the sperm tail
and to the developing sperm tail itself by intraflagellar transport. Outer dense fibers and a fibrous sheath appear shortly after the
axoneme completes its growth. The T-complex-associated–testis-expressed 1 (TCTE1) evolutionarily conserved axonemal protein
localizes to the nexin-dynein regulatory complex and is testis-enriched in its expression, with its mRNA appearing in early round
spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus.
Recent studies (Castanedaa et al., 2017) revealed that knockout of Tcte1 results in male sterility. Tcte1-null spermatozoa show aber-
rant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant
decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. As the authors note, these data
provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility as discussed
below.
 Spermatogenesis j Spermatids 45

Sperm Tail Enzymes

While structure and motility are relatively well-defined, the functions of the outer dense fibers and fibrous sheath are less clear. Most
of the glycolytic enzymes responsible for meeting sperm energy requirements are tightly bound to the fibrous sheath. These enzymes
include a sperm specific form of glyceraldehyde phosphate dehydrogenase (GAPDH-S) and lactate dehydrogenase A (LDHA). The
most abundant sperm specific protein, LDHC, is cytosolic and distributed throughout the sperm tail. Many species of spermatozoa,
including mouse and human, generate ATP by aerobic glycolysis to satisfy their energy requirements even though the mid-piece
houses the mitochondria that in most cells are the efficient energy producers. Both GAPD-S and LDHC have been proven to be
required for sperm function by targeted disruption of the cognate genes. When and how these sperm proteins become distributed
to the sperm tail is not obvious. For example, the Ldhc gene is transcribed and translated prior to and during meiosis, whereas Gapds
may be transcribed in spermatocytes but not translated until meiosis is completed. Clearly, it would be expected that the transcrip-
tional apparatus is disabled in haploid cells. To date, most of the testicular mRNAs known to be translationally regulated are
initially transcribed in postmeiotic cells. There are numerous studies in the last decade showing that mRNA is stored (often referred
to as “masked mRNA”) and is translated during spermiogenesis (Xu and Hecht, 2008). Additionally, there are examples of post-
transcriptional modifications of gene products during spermiogenesis that are beyond the scope of this article. Generally speaking
there are changes in transcription and translation throughout spermatogenesis, including spermiogenesis.

Mitochondria Migration

In elongating rodent and human spermatids, the mitochondria migrate toward the forming tail as the manchette appears (Fig. 1).
The mitochondria assemble along outer dense fibers, between the neck and the annulus, and assemble into a spiral structure in what
becomes the mid-piece of the sperm tail. The length of the sperm tail is species specific.

Intercellular Bridges

Intercellular bridges connect all the spermatids originating from the same stem cell, a consequence of the incomplete cytokinesis of
spermatogonia during mitosis and of spermatocytes during meiosis. Disruption of the intercellular bridges can cause male
infertility. The importance of intercellular bridges is clear. Thus, movement of molecules among haploid cells is made possible
by intercellular bridges, and this movement is likely to be involved in the sharing of mRNAs among germ cells. This sharing, in
turn, is important for sperm production because numerous essential proteins are encoded on sex chromosomes (Kim et al., 2015).

Spermiation

When the remodeling process of the spermatid has been completed, elongated spermatids are ready for release from the germinal
epithelium. The action of the steroid hormones estrogen and androgen, which are known to control transcription in various
reproductive and nonreproductive tissues, are important for the control of sperm release from the seminiferous epithelium
(spermiation). Spermiation is characterized by extensive remodeling of actin filaments and endocytosis. There is evidence suggest-
ing that genes regulated by estrogen and androgen may in some way be involved in actin remodeling and endocytosis.

Summary

This outline of spermiogenesis briefly describes the exquisitely complex structural and molecular intracellular changes required for
producing and shaping what is destined to become a functional male gamete. Other chapters will describe the involvement of Ser-
toli cell–germ ell interactions, and will provide further details about the contribution of Leydig cells in the interstitium. These inter-
actions too are essential for completing the highly-ordered process necessary for sperm production and thus survival of the species.

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