Ultrastructural study of spermatogenesis and sperm in the African medicinal leech Hirudo troctina Johnson, 1816 (Annelida, Hirudinida).

This paper presents the process of spermatogenesis in the leech Hirudo troctina Johnson, 1816 using light, fluorescent and transmission electron microscopy. At the onset of spermatogenesis in testes, the pear-shaped spermatogonia divide mitotically without full cytokinesis and as a result isogenic groups are formed (clusters, clones) with 2, 4, 8, 16, 32, 64, 128 spermatogonia and, finally, 256 primary spermatocytes occur. The final meiotic divisions of spermatocytes give rise to clones with 1024 spermatids. There are hundreds of developing germ-line clones in each testis. In each clone, the male germ cells divide in full synchrony and they are in the same phase of spermatogenesis. During complex spermiogenesis each spermatid becomes a filiform spermatozoon with a helicoid nucleus, which is characterized by the presence of a long acrosome with two regions - anterior and posterior, which are followed by a helicoid nucleus, a midpiece with only one mitochondrion and a long flagellum. Our results were compared to those on other clitellate annelids that have been studied to date, especially to sperm formation in Hirudo medicinalis Linnaeus, 1785. Only minor differences were found in the length and the diameter of different organelles and the number of spermatids in germ-line clones.

Deficiency in the omega-3 fatty acid pathway results in failure of acrosome biogenesis in mice.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.

Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats.

In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.

Haprin, a novel haploid germ cell-specific RING finger protein involved in the acrosome reaction.

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.

Ultrastructural changes induced by leaves of Azadirachta indica (Neem) in the testis of albino rats.

The present work was designed to study the effect of Azadirachta indica (Neem) powder on rat testis using the electron microscope. Male albino rats received 100 mg each A. indica leaf powder orally (by gavage). On alternate days, a second group of rats received 0.125 mg testosterone dipropionate intramuscularly. A third group received both A. indica leaf powder by gavage and testosterone dipropionate intramuscularly. Suitable controls were maintained. After autopsy, ultrastructural analysis of the testis revealed that animals treated with testosterone dipropionate showed well-developed Sertoli cells and germ cells with well-developed cytoplasmic organelles. By contrast, in A. indica-treated rats, intracellular spaces and vacuolization were observed in Sertoli cells; whereas in Leydig cells, cytoplasmic inclusions appeared diminished, and the configuration of granular endoplasmic reticulum appeared as a single unbranched tubule. In late spermatids, defects were observed in the mitochondrial sheath. The ultrastructural changes seen in the A. indica-treated group provide a clue that A. indica leaves might affect spermatogenesis through antispermatogenic and antiandrogenic properties.

Kinesin light-chain KLC3 expression in testis is restricted to spermatids.

Kinesins are tetrameric motor molecules, consisting of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs) that are involved in transport of cargo along microtubules. The function of the light chain may be in cargo binding and regulation of kinesin activity. In the mouse, two KLC genes, KLC1 and KLC2, had been identified. KLC1 plays a role in neuronal transport, and KLC2 appears to be more widely expressed. We report the cloning from a testicular cDNA expression library of a mammalian light chain, KLC3. The KLC3 gene is located in close proximity to the ERCC2 gene. KLC3 can be classified as a genuine light chain: it interacts in vitro with the KHC, the interaction is mediated by a conserved heptad repeat sequence, and it associates in vitro with microtubules. In mouse and rat testis, KLC3 protein expression is restricted to round and elongating spermatids, and KLC3 is present in sperm tails. In contrast, KLC1 and KLC2 can only be detected before meiosis in testis. Interestingly, the expression profiles of the three known KHCs and KLC3 differ significantly: Kif5a and Kif5b are not expressed after meiosis, and Kif5c is expressed at an extremely low level in spermatids but is not detectable in sperm tails. Our characterization of the KLC3 gene suggests that it carries out a unique and specialized role in spermatids.

Histochemical tracing of bismuth in testis from rats exposed intraperitoneally to bismuth subnitrate.

The histochemical silver amplification technique autometallography (AMG), was used to trace bismuth in the testis of Wistar rats injected intraperitoneally with bismuth subnitrate. In the seminiferous tubules, bismuth was located in lysosomes of Sertoli cells closely associated with heads of spermatids in the late stages of the spermatogenesis, i.e. shortly before the release of Step 19 spermatids in Stage XIII. No bismuth-specific AMG silver grains were detected in the spermatogenic cell line. However, tails of free sperm cells located in the tubular lumen showed autometallographic grains in close contact to the nine outer microtubule doublets in the axonema. Leydig cells concentrated huge amounts of AMG-bismuth in their lysosomes. Furthermore, parallel exposure to selenium significantly increased the amount of histochemically traceable bismuth in the rat testis.

Effect of Maytenus ilicifolia Mart.ex. Reiss on spermatogenesis.

The effect of the ethanolic extract of Maytenus ilicifolia Mart.ex. Reiss leaves on spermatogenesis was studied in Swiss mice by evaluating morphological characteristics by light and electron microscopy. The extract was administered at a dose of 200 mg/kg/day intraperitoneally for 20 days, and at a dose of 800 mg/kg/day orally for 30 days. Structural analysis of the germ epithelium showed that treated animals were not noticeably different from control animals. The alterations included some exfoliated immature germ cells, occasional germ cell death (recognized as pyknotic nuclei) and a few vacuolized seminiferous tubules. Ultrastructurally, enlarged lipid droplets were found in Sertoli cells and swollen acrosomes occurred in early spermatids of animals treated with the higher dose. Sperm production indicated that the ethanolic extract of M. ilicifolia leaves did not contain substances sufficient to arrest spermatogenesis.

PIP: Several plant extracts used to regulate female fertility have proved effective for the male reproductive system as well. Maytenus ilicifolia Mart.ex. Reiss, a plant native to parts of South America, has been used as a contraceptive, abortifacient, and emmenagogue by women in Paraguay and Argentina. The present study evaluated the effect of the ethanolic extract of Maytenus ilicifolia Mart.ex. Reiss leaves on spermatogenesis in Swiss mice. The extract was administered at doses of 200 mg/kg/day intraperitoneally for 20 days and 800 mg/kg/day orally for 30 days. Light microscopy revealed apparently normal spermatogenesis in the seminiferous tubules of treated animals. Although the spermatogenic process was not altered, ultrastructural alterations were observed, including some exfoliated immature germ cells, occasional germ cell death, and a few vacuolized seminiferous tubules. Enlarged lipid droplets were found in Sertoli cells and swollen acrosomes occurred in early spermatids of mice treated with the higher dose. However, sperm production indicated that the ethanolic extract did not contain substances sufficient to arrest spermatogenesis. Thus, the indigenous use of Maytenus ilicifolia as a medicinal plant probably does not cause a disturbance of spermatogenesis as a side effect.

An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Ultrastructural alterations in testes from rats treated with cysteine.

Cysteine administration in relatively large doses has been repetitively employed as preventive agent against chemically induced cell injury or as radioprotector. In this work we report that administration of a dose standard for those purposes (1.9 gr/kg, po in water) causes significant ultrastructurally evident alterations in testes at 24h. Damage involves Sertoli cells and spermatids. Alterations found in the former include dilatation of nuclear membrane and of the smooth (SER) and rough endoplasmic reticulum (RER) and detachment of ribosomes from RER. Cytoplasm appeared more sparse and electron lucent than in controls and contained more lipid droplets and lysosomes. Mitochondria exhibited alterations in shape and size. Damage in spermatids consisted of the appearance of irregular shape of the nucleus and alterations in their acrosomal caps. There was no histochemical evidence for either calcium accumulation or lipid peroxidation occurrence in testes of cysteine-treated animals. Results indicate that the large doses of cysteine employed in prevention of radiation or chemical effects is able to cause injury to Sertoli cells of the testes. Damage observed does not reach irreversible stages but may be sufficient to lead to production of abnormal spermatids.

Testicular changes in gonadotropin-independent familial male sexual precocity. Familial testotoxicosis.

A recently described form of male sexual precocity characterized by active Leydig cell differentiation and premature onset of spermatogenesis in the absence of pituitary gonadotropin stimulation has been termed familial testotoxicosis. The clinical and endocrine findings in the condition are consistent with an inherited intratesticular defect rather than central or true precocious puberty. In this report, testicular changes in biopsy specimens from a series of affected patients are presented. In all of the cases, Leydig cells demonstrated nuclear and cytoplasmic features characteristic of fully differentiated steroidogenic cells. Reinke crystals were absent. Germ cells at all stages of spermatogenesis were present, but there was evident disorganization of maturation. Spermatids exhibited a variety of structural abnormalities. Sertoli cells were characterized by complex cytoplasmic differentiation, Charcot-Böttcher crystals, and tight junction formation. The morphologic changes indicate premature differentiation of all of the major testicular cell types and are consistent with a distinctive type of intratesticular abnormality.

Gossypol--a new antifertility agent for males.

Gossypol is capable of inhibiting the fertility of male rats. On administration of gossypol the spermatids are damaged first. With the increase in dosage and duration of treatment the spermatocytes are also damaged. In the epidymis there are exfoliated spermatids and spermatocytes with numerous dead spermatozoa many of which had their heads and fails separated. The sperm count gradually decreases until azoospermia. Electron microscopic examinations reveal changes in the acrosomes and mitochondrial spiral sheath. Interstitial cells of the testis seemed to be unaffected. Over 4,000 healthy men have been on gossypol for more than 6 months. The antifertility efficacy evaluated by semen examination is 99.89%. Side effects are mild and of low incidence. A few of the subjects are found to be hypokalemic. However, it remains to be elucidated if gossypol is related to the development of hypokalemia.

Appearances of microtubules after various fixative procedures, and comparison with the appearances of tobacco mosaic virus.

Microtubules in crane-fly spermatids appeared altered when the glutaraldehyde-fixed cells were not postfixed with osmium tetroxide. The cytoplasmic microtubules were altered more than the doublet microtubules. Addition of osmium tetroxide after dehydration did not produce appearances identical with those of microtubules postfixed directly after glutaraldehyde, and thus at least some alterations occurred during dehydration, possibly due to extraction of microtubule-associated lipid. The omission of osmium tetroxide postfixation did not cause drastic alterations in the appearances of either tobacco mosaic virus (TMV), or polymerized tobacco mosaic virus protein (without RNA), suggesting that microtubule stability is different from TMV stability (with respect to the embedment procedure). The electron-dense stain associated with embedded-sectioned TMV is predominantly outside the TMV protein, as demonstrated by the known distribution of TMV protein compared with the dimensions of sectioned TMV and negatively stained TMV. The same might hold true for microtubules, as evidenced by the dimensions of negatively stained, isolated brain microtubules compared with those of embedded and sectioned brain microtubules.