Atlas of Clinical Andrology

Chapter 2. Functional ultrastructure of testis and epididymis

The genetic control of testis determination involves several genes: Tdy (testis determining factor), Sry (Y chromosome-specific gene), Sox6 (Sry-related gene), Sox9 (Sertoli cell-determining factor), TAZ83 (coding at early to mid-pachytene germ-cell stage), TAZ4 (testis-specific gene located on chromosome 11) and TNZ1 (expressed in neonatal Leydig cell) (Table 2.1).


The seminiferous epithelium, which lines the seminiferous tubules, is composed of two basic cell types: Sertoli cells and developing germ cells. The germ cells undergo a continuous series of cellular divisions and developmental changes, beginning at the periphery of the tubule and progressing towards the lumen. The stem cells, called spermatogonia, divide several times before becoming spermatocytes. The primary spermatocytes duplicate their DNA and undergo progressive nuclear changes during meiotic prophase known as preleptotene, leptotene, zygotene, pachytene and diplotene before dividing to form secondary spermatocytes, known as spermatocytogenesis. Without further DNA synthesis, the resultant secondary spermatocytes divide again to form the haploid cells known as spermatids (Figure 2.1). The spermatids then undergo a progressive series of structural and developmental changes to form spermatozoa.


The round spermatids are transformed into spermatozoa by a series of progressive changes collectively known as spermiogenesis. The changes include condensation of nuclear chromatin, formation of the sperm tail or flagellum apparatus, and development of the acrosomal cap. The various developmental stages of spermatic transformation are divided into four phases: Golgi body formation, cap formation, acrosomal and maturation phases. The reshaping of the nucleus and acrosome of each spermatid, initiated during the previous phase, produces the spermatozoon. Within the nucleus, the chromatin granules undergo progressive condensation as the transitional proteins are replaced by protamines which form a fine homogeneous material that uniformly fills the entire sperm nucleus.

Table 2.1 Genetic control of testis determination


Role during testis development

Tdy (testis determining factor in mice)

Act on supporting cell lineage and induce differentiation of supporting cells to Sertoli cells

Sry (a Y chromosome-specific gene)

Different homologs of Sry have a common open reading frame which has 41% homology to a DNA-binding motif HMG box; Sry encoded protein might have DNA-binding activity

Sox6 (Sry-related gene), Sox5


Overlapping functions in the adult mouse

A critical Sertoli cell differentiation factor


Coding at early to mid-pachytene germ-cell stage


Testis-specific gene located on chromosome 11


Expressed in neonatal Leydig cells

Figure 2.1 Spermatogenesis occurs within the seminiferous tubules of the testis, where all stages of sperm cell are nurtured by Sertoli cells.As spermatogenesis proceeds, the sperm cells migrate from the basal lamina to the lumen of the seminiferous tubule. Diploid spermatogonia proliferate mitot- ically to give rise to primary spermatocytes, and these divide meiotically to yield haploid secondary spermatocytes. The secondary spermatocytes undergo a further meiotic division and, hence, each primary spermatocyte gives rise to four round spermatids, each of which is destined to differentiate into a functional mature spermatozoon

During the later stages of spermiogenesis, the Sertoli cell shapes the cytoplasm remaining after elongation of the spermatid into a spheroidal lobule called the residual body. This lobule of cytoplasm, which remains connected to the elongated spermatid by a slender thread of cytoplasm, is also interconnected with other residual bodies by intercellular bridges that result from the incomplete division of the germ cells during spermatogenesis. Formation of the residual body completes the final maturation, and the elongated spermatids are ready for release as spermatozoa into the lumen of the seminiferous tubule (Figure 2.2).


Spermiation is the process by which the elongated spermatids, that are oriented perpendicularly to the tubular wall, are gradually extruded from the Sertoli cells into the lumen of the tubule. The lobules of residual cytoplasm, through which groups of spermatids are connected by intercellular bridges, remain embedded in the epithelium. Extrusion of the spermatozoa components continues until only a slender stalk of cytoplasm connects the neck of the spermatid to the residual body. Breakage of the stalk results in formation of the cytoplasm droplet in the neck region of the released spermatozoon (proximal droplet) and retention of the interconnected residual bodies. Following release of the spermatozoa the residual bodies are phagocytosed by the Sertoli cells to recycle the protoplasmic components. Not only do the Sertoli cells phagocytose the residual bodies remaining from the spermatogenic process, but they also remove considerable numbers of degenerated germ cells. Because the spermatogenic process is relatively inefficient, large numbers of potential sperm degenerate before maturation.

Figure 2.2 Spermiogenesis. Maturation of round spermatid into spermatozoa: (a) round spermatid, (b) elongating spermatid, (c) elongated spermatid and (d) spermatozoon. From Fleming and King (2003), with permission

Regulation of spermatogenesis

Spermatogenesis is a complex multistage process that involves formation of spermatogonia from germ stem cells, spermatogonial renewal and differentiation into primary spermatocytes, meiosis by which diploid spermatocytes develop into haploid spermatids, and spermiogenesis in which round spermatids mature into sperm. Spermatogenesis is a well-coordinated developmental program in which the different steps are defined by a cell type- and stage-specific induction or repression of the expression of specific genes. Many of these genes are expressed predominantly or exclusively in cells during spermatogenesis and their regulation can involve control at the transcriptional or post-transcriptional level. Various hormones, signaling pathways, transcription factors and interactions with the local environment play a critical role in regulating the different stages in this differentiation program. The identification of cell type- and stage-specific genes provides excellent tools to dissect the differentiation program and to study the mechanisms by which spermatogenesis is controlled. The germ cellspecific transcription factor cyclic adenosine monophosphate (cAMP) responsive element modulator (CREM) appears to be a key factor in the regulation of the expression of a number of postmeiotic genes. Several members of the nuclear receptor family (estrogen, retinoid and peroxisome proliferation activated receptors) are implicated in the regulation of specific stages of spermatogenesis.

The two compartments of the testes, the seminiferous tubules and interstitium, are under the hormonal control of gonadotropins, acting on either Sertoli cells or Leydig cells. During spermatogenesis, the different cell types and, in particular, germ cells express four specific sets of genes, which are regulated at either the transcriptional or transductional level. Some of these genes are detected exclusively in haploid cells, whereas others are initially expressed before or during meiosis and continue to be expressed in spermatids:

(1) Male germ cell-specific gene homologs (e.g. glyceraldehyde-3-phosphate dehydrogenase);

(2) Unique genes expressed exclusively during spermatogenesis;

(3) Germ cell-specific alternate transcripts leading to specific isoforms;

(4) Genes regulated developmentally during germcell differentiation (e.g. cAbl proto-oncogene).

Leydig cells also secrete non-pathogenic products, including growth factors, cytokines and vasoactive peptides, which contribute to the intercellular communication between Leydig, Sertoli and tubular cells. Testosterone initiates and maintains spermatogenesis.

Spermatogenic wave

The stages of the cycle within the seminiferous epithelium change not only with time, but also along the length of the tubular loop. At any one stage, a portion of tubule is adjacent to other portions of tubule in stages just preceding or following it in time. This sequential changing in cycle stage along the length of the tubule is known as the wave of the seminiferous epithelium. Examination of a loop of seminiferous tubule along its length also reveals that the wave involves a sequence of stages beginning with the less-advanced stages in the middle of the loop to progressively more-advanced stages nearer the rete testis (Figures 2.3-2.6Table 2.2).

Transferrin receptor

Intracytoplasmic sperm injection (ICSI) using testicular sperm is a valid option for the treatment of infertile couples in whom the male partner has azoospermia; however, testicular epididymal sperm extraction (TESE) may not be successful in all patients. Transferrin is the major secretory product of Sertoli cells, and is present in high concentrations in the seminal plasma of fertile men. Transferrin receptor is a transmembrane lipoprotein composed of two identical disulfide-linked 90-kDa subunits, providing iron for cell proliferation and differentiation. A soluble form of the transferring receptor (sTfr) can be detected in seminal plasma, probably originating from sper- matocytes/spermatids (El-Garem et al., 2003). Measurement of sTfr in the seminal plasma of patients with azoospermia may contribute to the etiological diagnosis of this condition. In combination with serum follicle stimulating hormone (FSH), this measurement may help to predict the outcome of TESE.

Seminal plasma

Primordial germ cells (PGCs) proliferate and migrate towards the genital ridges to become enclosed by Sertoli cells. These germ cells are then called gono- cytes. The proliferative activity of gonocytes is different from that of PGCs in that, after division, the daughter cells remain connected by an intercellular bridge. Moreover, gonocytes differ structurally from PGCs.

Gonocytes and A spermatogonia are not identified by their morphology. Hence, at the start of spermatogenesis germ cells called gonocytes, but as they develop further they are said to be a mixed population of As, Apr and Aal spermatogonia, and the more- differentiated Aj spermatogonia. Several researchers developed a monoclonal antibody to recognize a marker that is specifically expressed by the quiescent gonocytes until birth. It would appear that gonocytes are specific to fetal testes, whereas As, Apr and Aal spermatogonia of adult testes arise after birth. The development of gonocytes towards As, Apr and Aal spermatogonia, therefore, occurs during the quiescent period of germ cells, around the time of birth.

Most seminal plasma is produced by the prostate and seminal vesicles, while the testis and epididymis contribute less than 5% of the total semen volume. A low ejaculate volume reflects abnormality of the prostate or seminal vesicles. Fructose is produced by the seminal vesicles; thus the absence of fructose indicates either ejaculatory duct obstruction or seminal vesicle aplasia/hypoplasia. The latter can occur with cogenital bilateral absence of the vas deferens, which can be differentiated from ejaculatory duct obstruction by physical examination.

Blood-testis barrier

The seminiferous tubules are not penetrated by blood or lymph vessels. In addition, the developing germ cells within the tubules are protected from chemical changes in the blood by a specialized permeability barrier. This blood-testis barrier has two principal components: the incomplete or partial barrier of the myoid cells that surround the tubule; and the unique junctions between adjacent Sertoli cells.

In the testis, the blood-testis barrier (BTB) is located near the basal lamina, which effectively divides the epithelium into basal and adluminal compartments. During late stage VII through to early stage IX of the epithelial cycle, preleptotene and leptotene spermatocytes residing in the basal compartment adjacent to the basement membrane must traverse the BTB to gain entry to the adlu- minal compartment in order to develop into haploid spermatids. Without this timely movement of developing preleptotene and leptotene spermatocytes across the BTB, spermatogenesis is halted, leading to infertility.

Figure 2.3 Diagrammatic illustration of the cellular composition of the six stages of the cycle of the seminiferous epithelium in man.The stages are labeled (I)-(VI) and correspond to cell associations which succeed one another in time in any given area of the seminiferous tubule according to the sequence from left to right. After stage VI, stage I reappears and the sequence starts over again. The spaces allotted to the stages of the cycle are proportional to their relative durations. Ad, type A dark spermatogonium; Ap, type A pale spermatogonium; B,type B spermatogonium; R, preleptotene; L, leptotene; Z, zygotene; P, pachytene; Di, diplotene primary spermatocytes; Sptc Im, primary spermatocyte meiosis; Sptc II, secondary spermatocyte; Sa, Sb, Sc, Sd, spermatids; Ser, Sertoli cell (adapted from Hafez and Hafez, 2000)

Figure 2.4 Transmission electron microscopy of normal human testis. (a) Type A spermatogonium attached to tubular wall (W, arrow). Sertoli cell cytoplasm (S) expands between two primary spermatocytes (C) and spermatogonium (10200x). (b) Type B spermatogonium attached to basal lamina (arrow). Sertoli cell cytoplasm (S) interposed between this cell and primary spermatocyte (Cy) (6375x). (c) Primary spermatocyte: arrow points to short complex formed by sex chromosome cores (7800x). (d) Secondary spermatocyte endoplasmatic reticulum appears as flat saccules with localized dilatations, arranged concentrically around the nucleus (12000x). (e) Three maturing spermatids; condensing chromatin has ‘tigroid’ appearance (6000x). (f) Immature testis in a 4-year-old boy:gonocyte-like cell (G),transitional spermatogonium (T) and primitive (P) spermatogonium (1350x)

The BTB creates a unique microenvironment for germ cell development; it segregates immunologically most of the germ-cell antigens, except those residing on spermatogonia and preleptotene/ leptotene spermatocytes, from the systemic circulation, and maintains cell polarity. BTB dynamics in vitro and/or in vivo are regulated by cytokines, such as transforming growth factor (TGF)-03 and tumor necrosis factor (TNF)-a, via two defined signaling pathways:

(1) TGFp/MEKKs (MAP (mitrogen-activated protein)/ERK kinase kinase)/p38 MAP kinase;

(2) TNFa/integrin-linked kinase (ILK)/glycogen synthase kinase (GSK).

Figure 2.5 Stages of human spermatogenesis. Mature spermatozoa are obtained following mitotic, meiotic and post-meiotic phases. The cell types shown are spermatogonium type A dark (Ad-SG) and type A pale (Ap-SG), spermatogonium type B (B-SG), preleptotene (P1), leptotene (L), zygotene (Z) and pachytene (P) primary spermatocytes (IS), secondary spermatocytes (IIS), spermatids (S) at different stages of mutation (a, bl, b2, c, d) and mature spermatozoa with residual bodies (RB)

Figure 2.6 Seminiferous tubule showing Leydig and Sertoli cells

Table 2.2 Cytological characteristics of germinal epithelium


A spermatogonia

Attachment to basal lamina; oval nucleus with one or more corpuscles, finely granular chromatin that can be more deeply stained when nuclear ‘vacuole’ is present

B spermatogonia

Attachment to basal lamina can be complete, partial or almost absent round nucleus with fine chromatin; heterochromatic granules and one or two less conspicuous nucleoli

Primary spermatocytes

No attachment to basal lamina; nucleus with filamentous (leptotene or thick pachytene chromosomes;the sexual pair can appear to be heterochromatic (‘sex vesicle’); pachytene spermatocytes with large nucleoli are large germ cells

Secondary spermatocytes

Smaller nucleus, fine chromatin; similar to those of young spermatids although larger; infrequently recognized


Round nucleus with fine chromatin or small, elongated, deeply chromatic nucleus; presence of acrosome granule or head cap, both PAS positive; flagellum soon becomes evident, as well as ‘manchette’; when mature forms are free in lumen (spermatozoa), discarded cytoplasms appear as residual bodies

Sertoli cell

Large cell with numerous extensions between germ cells; nucleus large and polymorphous, showing infoldings of nuclear membrane; large nucleolus with eosinophilic core



Very large cell with basophilic cytoplasm situated in core of germinal cord without attachment to basal lamina; degeneration frequently seen; disappearing progressively after birth

Intermediate type

Large cells, similar to gonocytes but showing attachment to basal lamina; gradually disappearing during infancy

Primitive type spermatogonia

Smaller oval cell with light basophilic cytoplasm; round nucleus with fine chromatin showing 1 or 2 nucleoli; practically the only genial type when puberty starts

Immature supporting cell

Scarce cytoplasm with poorly defined limits; elongated nucleus with palisade-like arrangement; 1 or 2 small nucleoli; frequently dividing during infancy

PAS, periodic acid-Schiff

Myoid layer

The basement membrane or tunica propria that surrounds the seminiferous tubules contains a layer of contractile myoid cells. In some species the majority of the cell junctions of this layer are sealed by tight apposition of the adjacent cell membranes. This barrier, however, is not well developed in the bull, ram or boar and may be relatively unimportant in the testis of farm animals.

Sertoli cell junctions

The principal permeability barrier between the blood and testis is thought to be the complexes at junctions between adjacent Sertoli cells (Figures 2.7 and 2.8). These Sertoli-Sertoli junctions, which are situated near the cellular base, contain multiple zones of adhesion (tight junctions) where the opposing membranes are fused. They consist of a basal compartment containing spermatogonia and preleptotene spermatocytes, and an adluminal compartment containing the more advanced stages of spermatocytes and spermatids, which freely communicates with the lumen of the tubule.

Nitric oxide and nitric oxide synthase

Nitric oxide (NO) and nitric oxide synthase (NOS) play a pivotal role in epithelial barrier function in many organs, including the testis, and in spermatogenesis. BTB function is maintained by intricate regulatory mechanisms. In addition to hormones and cytokines, nitric oxide (NO) is a putative tight junction regulator in the testis; tight junction dynamics are regulated by three tight junction integral membrane proteins via three possible signaling pathways.

Figure 2.7 Nitric oxide (NO)/nitric oxide synthase (NOS) and tight junctions in the testis.The three tight junction-integral membrane proteins (JAM, occludin and claudin) are shown at the site of the blood-testis barrier, as well as the three possible signaling pathways (I-III) that regulate the opening and closing of the Sertoli cell tight junctions. (I) NO stimulates soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP), leading to tight junction disruption.The cGMP can also activate protein kinase G (PKG), which, in turn, can affect tight junction dynamics via its effects on occludin, reducing the level of occludin at the site of Sertoli cell tight junctions, thereby opening up the tight junction barrier. (II) Tumor necrosis factor-а (TNFa) activates the integrin-linked kinase (ILK)/glycogen synthase kinase (GSK)/p130cas/c-Jun N-terminal kinase (JNK) signaling pathway which, in turn, affects the level of occludin.Transforming growth factor-β3 (TGFβ3) activates mitogen-activated protein (MAP)/ERK kinase kinase 2 (MEKK2) and p38 MAP kinase to perturb Sertoli cell tight junctions via its effects on the level of occludin and zonula occudens (ZO)-1 at the site of the blood-testis barrier. BTB, blood-testis barrier; TIMP-1, tissue inhibitor of metalloproteinase-1; MMP-9, matrix metalloproteinase-9; MAGI, membrane-associated guanylate kinase with inverted orientation. From Lee and Cheng (2004), with permission

Figure 2.8 Nitric oxide (NO) and nitric oxide synthase (NOS) effects on tight junctions in the testis. A low concentration of NO (< 1 |lmol/l) can stimulate other cellular downstream signaling pathways. In addition, NO can activate soluble guanylate cyclase (sGC) and adenylate cyclase (AC) to synthesize cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), respectively.The cAMP activates protein kinase (PKA). Likewise, the cGMP has more diverse effects by activating phosphodiesterase (PDE), cyclic nucleotide-gated channel (CNG) and PKG. Both cAMP and cGMP are known regulators of Sertoli cell tight junction (TJ) dynamics. PDE can negatively control the levels of cAMP and cGMP. In addition, NO can stimulate mitogen-activated protein (MAP) kinases, such as p38, c-Jun N-terminal kinase (JNK) and MAP/ERK kinases 1 and 2 (MEK 1/2). In the testis, cAMP/PKA and cGMP/PKG are putative regulators of tight junction dynamics. DHT, 5a-dihydrotestosterone; EGF, epidermal growth factor; LPS, lipopolysaccharides; VEGF, vascular epithelial growth factor; T, testosterone; GFs, growth factors; IL, interleukin. From Lee and Cheng (2004), with permission

Table 2.3 Epididymal sperm maturation, capacitation and acrosome reaction

Maturational stage


Sperm gain fertilization potential

Ability of testicular sperm to move is due to immaturity of plasma membrane

Development of sperm motility

Transfer of substances: glycerol-3-phosphorylcholine a forward motility protein from the epididymal fluid

Maturation of sperm plasma

Sperm unable to move progressively or interact with and fertilize eggs


Sperm attain motility in caput epididymis or corpus epididymis according to species

Maturational sperm structures

Sperm do not gain fertilization potential simultaneously in same region

Most sperm attain full fertilization potential in cauda, major sperm storage site

Deposit, storage, ascent in female tract

Sperm fertilization potential is evaluated by: (1) sperm’s ability to fertilize zona-free hamster eggs; (2) motility patterns; (3) surface characteristics; and (4) structural stability of head/tail

Epididymis/vas deferens secrete specific components necessary for functional maturation

Two other tight junction-integral membrane proteins, namely CAR and CRBI, are found in other epithelia. Sertoli cell tight junctions that constitute the BTB physically divide the seminiferous epithelium into adluminal and basal compartments. Underneath the basal compartment is the tunica propria, which is composed of a non-cellular and a cellular zone. The cellular zone includes the basement membrane. In the testis, the basement membrane is a modified form of extracellular matrix composed largely of type IV collagen, laminin, heparin sulfate proteoglycan and entactin, which is adjacent to the seminiferous epithelium; behind this is a layer of type I collagen fibrils. The cellular zone is made up of a layer of myoid cells; behind this lies the lymphatic endothelium. The three classes of tight junction-integral membrane proteins, namely occludins, claudins and JAMs, interact with adaptors, such as zonula occludens (ZO)- 1, ZO-2, afadin, cingulin and membrane-associated guanylate kinase with inverted orientation (MAGI), tethering actin filaments to the tight junction-integral membrane proteins and recruiting proteins to the BTB site (Figure 2.7).


Extensive investigations have been conducted on phagocytosis, which is an oxygen-dependent process. During non-specific immunological defense, the activity of pentose phosphate is dramatically increased leading to the formation of reduced nicotinamide adenosine dinucleotide phosphate (NADPH). NADPH is also generated in leukocytes by oxidation of glucose-6-phosphate. As NADPH is needed to reduced the oxygen bound to membrane-based cytochromes, the oxygen demand is strongly increased (respiratory burst). During this process oxygen is converted into superoxide anions, hydrogen peroxide, monomolecular oxygen and hydroxyl radicals, which are effective microbicidals, by several types of phagocytic cells (neutrophils, eosinophils, basophils and macrophages).


During epididymal maturation, membrane lipids in the sperm undergo distinct physical and chemical alterations, including changes in the distribution pattern of intramembranous protein (lipoprotein) particles in the sperm plasma membrane of epididymal sperm (Table 2.3). The plasma membrane contains both membrane-integrated and surface-adsorbed proteins. Some of these intrinsic proteins change their location in or on the plasma membrane during sperm maturation. These dynamic changes occur throughout the male reproductive tract, but most actively in the caput and corpus epididymis leading to the sperm becoming fertilizable. During the maturation of epididymal sperm, some surface lipoprotein, either membrane-integrated or membrane-adsorbed, are located over the entire sperm head. Other proteins are restricted to the acrosomal or postacrosomal region of the head (Table 2.4). These lipoproteins/ polypeptides stabilize the plasma membrane and prevent premature acrosome reactions. Maturational interactions of epididymal sperm are not limited to those involving the sperm head; adsorption and/ or integration of several specific glycoproteins and peptides occurs on or in the plasma membrane of the tail (Figures 2.9-2.11).

Figure 2.9 Principal cell of the caput epididymis on the left and of the corpus epididymis on the right, with a clear cell in between, as visualized by electron microscopy. A halo cell and a basal cell are also shown. Principal cells of both regions contain coated pits (cp), endosomes (E), lyosomes (L) and elaborate Golgi apparatus (G). Rough endoplasmic reticulum (rER) occupies the basal region of the principal cell of the caput, while numerous lipid droplets (Lip) occupy the cytoplasm of the principal cells of the corpus region. The clear cell shows few microvilli (MV), but numerous coated pits, small apical vesicles (v), endosomes and lysomes, all of which are involved in endocytosis.The halo cell is inserted between adjacent principal cells, located basally, and contains small dense core granules (g), while the basal cell stretches inself along the basement membrane (BM). M, mitochondria; N, nucleus. From Robaire (2002), with permission

Figure 2.10 Age-related changes in the expression of E-cadherin, occludin and zonula occludens (ZO)-1,as well as in the permeability of lanthanum in the Brown Norway rat corpus and cauda epididymis. From Robaire (2002), with permission

Figure 2.11 (a) Luminal surface of an efferent duct with ciliated and non-ciliated cells and a spermatozoon. (b) Short microvilli of the luminal surface of non-ciliated cells in the efferent ducts.The cytoplasm droplet (CD), acrosome (A) and middle piece (MP) of the spermatozoon are distinguishable by scanning electron microscopy (6500x). (c) Cross-section of the distal cauda epididymis. Thick layers of smooth muscle surround the highly infolded epithelium (25x). (d) Sagittal section of the corpus epididymis. Columnar cells covered with stereo cilia. Undulations are due to differences in cell height (276x). From Johnson (1991), with permission

Figure 2.12 Scanning electron microscopy of epididymal sperm, with cytoplasm droplets (arrows), at different stages of immaturity in different segments of the epididymis

Sperm maturation in the epididymis is associated with complex biochemical, biophysical and ultrastructural molecular changes including loss of total protein, increase in cAMP, extensive modifications of energy metabolism, changes in lipoprotein content, changes in size, shape and internal structure of acrosome, variations in cohesiveness, migration of cytoplasm droplets from outer acrosomal membranes (Figures 2.11 and 2.12), increased disulfide bonds in the sperm tail with motility changes, alterations in antigenic properties of sperm surface and a net increase in negative surface charge, possibly due to addition of sialic acid groups.

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