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Spermiogenesis is a long process whereby haploid spermatids derived from the meiotic divisions of spermatocytes undergo metamorphosis into spermatozoa. It is subdivided into distinct steps with 19 being identified in rats, 16 in mouse and 8 in humans. Spermiogenesis extends over 22.7 days in rats and 21.6 days in humans. In this part, we review several key events that take place during the development of spermatids from a structural and functional point of view. During early spermiogenesis, the Golgi apparatus forms the acrosome, a lysosome-like membrane bound organelle involved in fertilization. The endoplasmic reticulum undergoes several topographical and structural modifications including the formation of the radial body and annulate lamellae. The chromatoid body is fully developed and undergoes structural and functional modifications at this time. It is suspected to be involved in RNA storing and processing. The shape of the spermatid head undergoes extensive structural changes that are species-specific, and the nuclear chromatin becomes compacted to accommodate the stream-lined appearance of the sperm head. Microtubules become organized to form a curtain or manchette that associates with spermatids at specific steps of their development. It is involved in maintenance of the sperm head shape and trafficking of proteins in the spermatid cytoplasm. During spermiogenesis, many genes/proteins have been implicated in the diverse dynamic events occurring at this time of development of germ cells and the absence of some of these have been shown to result in subfertility or infertility., (Copyright 2009 Wiley-Liss, Inc.)

Spermatogenesis, a study of germ cell development, is a long, orderly, and well-defined process occurring in seminiferous tubules of the testis. It is a temporal event whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa over a period of several weeks. Spermatogenesis is characterized by three specific functional phases: proliferation, meiosis, and differentiation, and it involves spermatogonia, spermatocytes, and spermatids. Germ cells at steps of development form various cellular associations or stages, with 6, 12, and 14 specific stages being identified in human, mouse, and rat, respectively. The stages evolve over time in a given area of the seminiferous tubule forming a cycle of the seminiferous epithelium that has a well-defined duration for a given species. In this part, we discuss the proliferation and meiotic phase whereby spermatogonia undergo several mitotic divisions to form spermatocytes that undergo two meiotic divisions to form haploid spermatids. In the rat, spermatogonia can be subdivided into several classes: stem cells (A(s)), proliferating cells (A(pr), A(al)), and differentiating cells (A(1)-A(4), In, B). They are dependent on a specific microenvironment (niche) contributed by Sertoli, myoid, and Leydig cells for proper development. Spermatogonia possess several surface markers whereby they can be identified from each other. During meiosis, spermatocytes undergo chromosomal pairing, synapsis, and genetic exchange as well as transforming into haploid cells following meiosis. The meiotic cells form specific structural entities such as the synaptonemal complex and sex body. Many genes involved in spermatogonial renewal and the meiotic process have been identified and shown to be essential for this event., (Copyright 2009 Wiley-Liss, Inc.)

As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic., (Copyright 2009 Wiley-Liss, Inc.)

Huntingtin interacting protein 1 (HIP1) is an endocytic adaptor protein with clathrin assembly activity that binds to cytoplasmic proteins, such as F-actin, tubulin, and huntingtin (htt). To gain insight into diverse functions of HIP1, we characterized the male reproductive defect of HIP1(-/-) mice from 7 to 30 weeks of age. High levels of HIP1 protein were expressed in the testis of wild-type mice as seen by Western blots and as a reaction over Sertoli cells and elongating spermatids as visualized by immunocytochemistry. Accordingly, major structural abnormalities were evident in HIP1(-/-) mice with vacuolation of seminiferous tubules caused by an apparent loss of postmeiotic spermatids and a significant reduction in mean profile area. Remaining spermatids revealed deformations of their heads, flagella, and/or acrosomes. In some Sertoli cells, ectoplasmic specializations (ES) were absent or altered in appearance accounting for the presence of spherical germ cells in the epididymal lumen. Quantitative analyses of sperm counts from the cauda epididymidis demonstrated a significant decrease in HIP1(-/-) mice compared to wild-type littermates. In addition, computer-assisted sperm analyses indicated that velocities, amplitude of lateral head displacements (ALH), and numbers and percentages of sperm in the motile, rapid, and progressive categories were all significantly reduced in HIP1(-/-) mice, while the numbers and percentages of sperm in the static category were greatly increased. Taken together, these various abnormalities corroborate reduced fertility levels in HIP1(-/-) mice and suggest a role for HIP1 in stabilizing actin and microtubules, which are important cytoskeletal elements enabling normal spermatid and Sertoli cell morphology and function., ((c) 2006 Wiley-Liss, Inc.)

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) is a multifunctional fatty acyl esterase that is essential for male fertility and spermatogenesis and that also plays important roles in the function of adipocytes, pancreatic beta-cells, and adrenal cortical cells. Gene-targeted HSL-deficient (HSL-/-) male mice are infertile, have a 2-fold reduction in testicular mass, a 2-fold elevation of the ratio of esterified to free cholesterol in testis, and unique morphological abnormalities in round and elongating spermatids. Postmeiotic germ cells in the testis express a specific HSL isoform. We created transgenic mice expressing a normal human testicular HSL cDNA from the mouse protamine-1 promoter, which mediates expression specifically in postmeiotic germ cells. Testicular cholesteryl esterase activity was undetectable in HSL-/- mice, but in HSL-/- males expressing the testicular transgene, activity was 2-fold greater than normal. HSL transgene mRNA became detectable in testes between 19 and 25 days of age, coinciding with the first wave of postmeiotic transcription in round spermatids. In contrast to nontransgenic HSL-/- mice, HSL-/- males expressing the testicular transgene were normal with respect to fertility, testicular mass, testicular esterified/free cholesterol ratio, and testicular histology. Their cauda epididymides contained abundant, normal-appearing spermatozoa. We conclude that human testicular HSL is functional in mouse testis and that the mechanism of infertility in HSL-deficient males is cell autonomous and resides in postmeiotic germ cells, because HSL expression in these cells is in itself sufficient to restore normal fertility.

Transition proteins replace testis-specific histones and are finally replaced by protamines in the nucleus of germ cells during spermiogenesis. In this study, immunoperoxidase and immunogold localization were used to determine both qualitatively and quantitatively the intracellular distribution of testis-specific histone (H1t), transition protein 1(TP1), and transition protein 2 (TP2) during rat spermatogenesis. H1t labeling was concentrated over heterochromatin in the nucleus of late-pachytene spermatocytes and spermatids up to mid-steps 10. In step 9 spermatids, H1t was confined to the caudal end of the nucleus where heterochromatin was still present, while in early step 10 spermatids, only a few of the nuclei remained caudally labeled. In late step 10 spermatids, a fibrillar chromatin network was distributed throughout the nucleus coincident with the loss of H1t. A statistically significant rise in TP1 and TP2 labeling density over control values was first encountered in the nucleus of step 11 spermatids coincident with the initiation of condensation of the fibrillar chromatin. The TP1 and TP2 labeling density progressively increased in nucleus of step 11-13 spermatids with the apical to caudal condensation of the fibrillar chromatin, In step 13 spermatids, the chromatin was homogeneously condensed throughout the nucleus. In the case of TP1, the nuclear labeling density gradually declined after step 13 and disappeared by step 17. In the case of TP2, the nuclear labeling density disappeared by step 16. This study shows that, coincident with the loss of H1t, the chromatin of the spermatid is reorganized into a fibrillar network, whereas, coincident with the appearance and progressive increase of TP1 and TP2, the fibrillar chromatin condenses in an apical to caudal direction in the nucleus of the spermatid. Thus the remodeling of chromatin structure during spermiogenesis appears to be a two-step process that is sequentially influenced by the loss of spermatid-specific histones and the appearance of transition proteins.

Background: The exact structural relationships of the saccules, membranous tubules, and vesicles that compose the cis- and mid-compartments of the Golgi cortex of rat spermatids was investigated to determine the relationship of these elements to each other., Methods: Tissues fixed with glutaraldehyde and buffered in sodium cacodylate were examined with the electron microscope. Electron micrographs, including stereopairs, were analyzed to determine the three-dimensional organization of the Golgi elements., Results: The deeper layer of the Golgi cortex was composed of stacks of saccules connected to each other either by saccules or membranous tubules. The peripheral region of the Golgi cortex, located between the cis-side of the stacks and a network of overlying ER cisternae contained numerous membranous tubules and vesicles of two class sizes: 50-100 nm vesicles and microvesicles 5-10 nm in diameter. The tubules formed tight networks, known as cis-elements or cis-Golgi networks (CGN), which were strictly parallel and next to the first or cis-saccule of the stack. The cis-elements were continuous with more loosely arranged peripheral tubules which formed elaborate, intertwined and interconnected networks. These peripheral tubules closely approximated the overlying ER cisternae in areas often showing fuzz-coated finger-like projections. Occasionally such peripheral tubules were continuous with ER cisternae. The saccules forming the stacks were continuous with membranous tubules which not only connected saccules of adjacent stacks, but also saccules of the same stack. These tubules were also connected with the tight tubular networks forming the cis-elements and the broad networks formed by the peripheral membranous tubules. Vesicles (50-100 nm) and microvesicles (5-10 nm) frequently formed aggregates in the peripheral Golgi region next to areas of ER membrane free of fuzz-coated projections. The microvesicles, embedded in a denser cytoplasmic matrix, had a more or less distinct delimiting membrane suggestive of their disintegration in this juxta-ER location. The 50-100 nm vesicles that were seen at the periphery of the vesicular aggregates appeared to form mainly from the membranous tubules of the Golgi cortex., Conclusions: Thus the saccules and membranous tubules of the spermatid's Golgi cortex formed a single continuous membraneous system connected to ER cisternae. The vesicles, seemingly arising from the membranous tubules, appear to follow a retrograde pathway and undergo dissolution next to ER cisternae.

Recently, a cDNA that coded for an enteric smooth muscle gamma-actin (SMGA) that was expressed in post-meiotic mouse testicular cells was identified. To determine the cellular location(s) of the protein encoded by this cDNA, this SMGA was probed for by immunocytochemistry in the cells of the seminiferous epithelium with two different monoclonal antibodies (Mabs), B4 and HUC 1-1, known to be muscle actin selective. As a control, we also examined the immunoreactivity of a third Mab, C4, that reacts with all non-muscle and muscle vertebrate isoactins. Using light and electron microscopy, a progressive increase in immunolabeling was observed with the muscle selective HUC 1-1 Mab over a loose actin filamentous network distributed throughout the cytoplasm of steps 4-16 spermatids. Thereafter, the labeling decreased such that at step 17 spermatids, only cytoplasmic labeling in the tail of the spermatids was observed. No labeling of this network was noted with the C4 or B4 Mabs. However, myoid cells enveloping seminiferous tubules and smooth muscle cells of interstitial blood vessels demonstrated comparable intense labeling with each of the three Mabs. The C4 Mab intensely labeled actin filaments of the Sertoli-Sertoli and Sertoli-spermatid ectoplasmic specializations. Also well labeled were numerous actin filaments found in the apical Sertoli cell processes encapsulating the heads of late step 19 spermatids at stage VII of the cycle of the seminiferous epithelium. In addition, actin filamentous bundles enveloping tubulobulbar complexes of the late spermatids within the Sertoli cell apical processes were intensely labeled. The actin filaments in the Sertoli apical processes and surrounding the tubulobulbar complexes were also strongly immunolabeled with the HUC 1-1 Mab. The C4 Mab but not the B4 or HUC 1-1 Mabs, recognized actin in the subacrosomal space of steps 4-18 spermatids. This study suggests that there are muscle isoforms of actin within the cytoplasm of developing spermatids and within apical processes of Sertoli cells.

Affinity purified antibodies prepared against proteins isolated from fibrous sheath (FS) and outer dense fibers (ODF) were utilized in an immunocytochemical study of spermatids at various steps of spermiogenesis. This study, using the immunogold technique, was performed on sections of Epon or Lowicryl embedded tissues examined with the electron microscope. In the case of FS antibodies there was a selective immunoreactivity of the FS itself from step 10 onwards, but no reactivity over the plasma membrane associated FS anlagen. In addition there was a diffuse immunoreaction over the cytoplasmic matrix from step 9 until step 18 of spermiogenesis but no reactivity over the various types of dense bodies (e.g., granulated bodies, reticulated body, etc.) seen in the cytoplasm of these spermatids. In the case of ODF antibodies the ODF were immunolabeled throughout their development from step 11 onward. In addition to a diffuse immunoreactivity of the cytoplasmic matrix of spermatids from step 9 until step 18 of spermiogenesis, there was an immunolabeling of "granulated bodies." These bodies appeared in relation to ER cisternae during steps 10-14, increased in number and size during steps 15-17 and decreased in number thereafter leaving only a few coarsely granulated bodies in the residual cytoplasm which detached from late step 19 spermatids. No other cytoplasmic structures were labeled with the ODF antibody-gold complexes. Thus the granulated bodies appeared to serve as a transitory storage site for some proteins destined to form ODF, a major cytoskeletal element of the tail of rat spermatozoa.

Throught stage VII and early stage VIII of the cycle of the seminiferous epithelium, the heads of the late spermatids, located in a juxtaluminal position, are embedded in apical processes of Sertoli cells. These processes contain cisternae of endoplasmic reticulum (ER) of two main types, i.e., flattened and tubular, which communicate with each other to form a continuous system. Throughout the long stage VII of the cycle, these two types of cisternae undergo marked changes. In early stage VII, the flattened cisternae, developing from the subsurface cisternae which compose the "junctional specialization," form concentric sheets at the periphery and in the middle of each apical process. The less conspicuous tubular cisternae form a continuous network which is present in the bridge connecting the Sertoli cell body to the apical process, and extends along the dorsal and ventral aspects of the spermatid's head to end up as cup-shaped flattened cisternae capping the bulbs of the tubulobulbar complexes described by Russell and Clermont ('76). In mid stage VII, the flattened cisternae start to regress, while the tubular cisternae become more abundant. In late stage VII, only fragments of the flattened cisternae are present, while the tubular cisternae form a profuse and elaborate network throughout the apical process. In the following stage VIII, the tubular cisternae disperse and only remnants of ER are present at the time of the release of the spermatid into the tubular lumen. These transformations of ER cisternae suggest a complex alteration in the relationship between Sertoli cells and late spermatids prior to their release as spermatozoa.

Glutaraldehyde-fixed testes were impregnated with the Ur-Pb-Cu technique of Thiéry and Rambourg ('76) or postfixed in ferrocyanide-reduced osmium (Karnovsky, '71). Thin and thick (0.5 micron) sections were examined with a Philips 400 electron microscope at 80 or 100 kv. Stereopairs were prepared from pictures of the same field after tilting the specimen every 6 degrees from the -45 degree to the +45 degree position of EM goniometric stage. The cortex of the compact hemispherical Golgi apparatus of young spermatids (steps 2-8) was found to be composed of saccular and intersaccular regions similar to those described in the Golgi apparatus of Sertoli cells (Rambourg et al., '79). In the saccular region, the stacks were composed of three to nine parallel saccules perforated with pores of various dimensions. On the mature or trans-face of the stack, one or two membranous elements with a wider lumen were either closely applied to the overlying saccules or were separated from them and intermixed with the vesicular components of the medulla. On the forming or cis-face of the stack, three or four saccules were frequently interrupted by gaps in register from one saccule to another. In three dimensions, these gaps appeared as pan-shaped spaces or "wells," often containing a few vesicles. Immediately overlying the first saccule on the cis-face, a regular network of anastomotic tubules was present, corresponding to the cis-osmiophilic element observed in other cell types. In the intersaccular region, membranous tubules connected to the edges of the saccules branched, intertwined, anastomosed, and bridged adjacent stacks of saccules. Such membranous tubules bridged saccules with the cis-osmiophilic element or saccules of the same stack. Between the ER cisternae capping the surface of the Golgi apparatus and the cis-network of anastomotic tubules, there was a space called the peripheral Golgi region containing small vesicles and membranous tortuous tubules. The vesicles were frequently arranged in clusters that were capped by an ER cisterna and displayed a size gradient from the periphery to the center of the cluster. Thus, although there were similarities between the three-dimensional architectures of the Golgi apparatus in Sertoli cells and young spermatids (e.g., saccular and intersaccular regions), several structural features distinguished the spermatid's Golgi apparatus.

The "round body," a spherical structure typically associated with a nucleolus in male germ cells of the rat, has been examined in the electron microscope using routine and cytochemical methods to determine its structure, composition, and mode of development. Cytochemical analysis indicates that the round body includes neither nucleic acid nor lipid, but is composed of nonhistone protein which appears in the form of 1.6-nm-wide fibrils. Development begins in late leptotene, when a single round body appears in each spermatocyte as an irregular spheroid located along the inner surface of the nuclear envelope. During subsequent stages of the meiotic prophase, the round body leaves the nuclear envelope, becomes a regular sphere, and gradually enlarges from a diameter of 0.4 micron in leptotene to 1.6 micron in diplotene. Concurrently, lacunae appear within its substance and enlarge. At each maturation division, the amount of round-body material is decreased by about half, presumably because the constituent proteins are dissociated at metaphase, distributed between the two daughter cells at telophase, and reconstituted into half-sized round bodies. As spermiogenesis proceeds, the round body shrinks gradually and disappears at step 8. Soon after its appearance at leptotene, the round body becomes associated with and is surrounded by the pars granulosa of one of the nucleoli. Moreover, 3H-uridine incorporation into nucleolar RNA is high as long as the size of the round body increases, but is low or absent when it decreases. It is possible, therefore, that the round body exerts some control on nucleolar activity in meiotic cells.

The cytochemical distribution of two different acid phosphatases, nicotinamide adenine dinucleotide phosphatase (NADPase) and cytidine monophosphatase (CMPase), was analyzed within the Golgi apparatus, acrosomic system, and chromatoid body of early spermatids. Within the saccules composing the Golgi cortex, the phosphatases were localized in specific and nonoverlapping compartments: the cis-tubular network, or cis-element, and the first underlying saccule were unreactive for both enzymes, the following four to five saccules were reactive for NADPase, and the last three saccules were reactive for CMPase. Vesicles of various sizes and short membranous tubules, present near the edges of saccules and in the medulla of the Golgi apparatus, were NADPase- and/or CMPase-positive. The acrosomic system showed a weak but significant NADPase reactivity and a strong CMPase reactivity. The vesicles associated with the chromatoid body were reactive for NADPase and/or CMPase. The combined observations indicate that the two cytochemically distinct compartments of the Golgi cortex, the NADPase- and CMPase-positive saccules, independently contribute to the formation of the acrosomic system and the vesicular component of the chromatoid body.