Acrosome Phase Research Articles

Development of membrane differentiations in the guinea pig spermatid during spermiogenesis.

AbstractWe have studied the development of membrane differentiations in guinea pig spermatids during spermiogenesis, using electron microscopy of thin sections, freeze‐fracturing, and filipin labeling as an indicator of membrane cholesterol distribution. The development of the distinctive membrane specializations closely correlated with the developmental steps of spermatid differentiation. The annulus, the zipper, and the circumferential strands of particles overlying the mitochondrial gyres of the midpiece appeared in the plasma membrane over the tail during the last two steps of spermiogenesis. The outer acrosomal membrane showed trnsient crenations during the acrosome phase and serrations over the equatorial segment of the acrosome during the maturation phase. On the inner nuclear membrane, a depression appeared opposite the inner zone of the acrosome during the Golgi phase and persisted throughout spermiogenesis. The implantation fossa also appeared during this phase, as a bulging on the inner nuclear envelope covered with small membrane particles. By the cap phase, small and large 15–20‐nm particles were present at the implantation‐fossa site, and clusters of 9‐nm particles were seen over the postacrosomal segment of the nuclear envelope. The migration of the nuclear pores began when close contacts were established between adjacent nuclear membranes. It started simultaneously at the anterior pole and the implantation fossa and later progressed over the segment of membrane between the caudal margin of the acrosome and the posterior ring. By the maturation phase, the nuclear pores had migrated to the redundant nuclear envelope.After filipin labeling, a gradient was visible in the distribution of the cholesterol‐filipin complexes, decreasing from the plasma membrane to the nuclear membrane. Within the same membrane, cholesterol distribution varied from one pole of the cell to the other. On the differentiations of the plasma membrane over the tail, cholesterol‐filipin complexes were absent, but they were profuse in the membrane over the head. In the acrosomal membrane, the complexes were gathered in clumps associated with the crenations. They became fewer in the rostral segment but remained moderate in the caudal equatorial segment as spermiogenesis proceeded. The nuclear membrane contained cholesterol‐free regions opposite the inner acrosome and at the site of the implantation fossa. Otherwise, the acrosomal segment of the nuclear envelope demonstrated a homogeneous distribution of cholesterol‐filipin complexes, while the postacrosomal segment showed small clumps of complexes adjacent to nuclear pores. We propose that each of the spermatid cell membranes is not an autonomous system, but is dependent upon its exchanges with the immediate external environment under the one side of the membrane and its close coupling to the internal environment (i.e., membrane‐associated structures: annulus, zipper, etc.) under the otehr side of the membrane. We suggest that the cholesterol in spermatid cell membranes contributes to the establishment of their mosaicism and regulates the modeling of the spermatid by modulating the internal fluidity of individual membrane segments during spermiogenesis.

Ultrastructural changes in the spermatid nucleolus during spermiogenesis in the guinea pig

AbstractMorphological changes in the ring‐shaped nucleoli of spermatids were observed by electron microscopy during spermiogenesis in the guinea pig. In the early acrosomal phase each nucelolus was composed of a bundule of fibers about 25 nm in diameter and 250 nm in length. In cross section the fibers were seen to be closely packed in hexagonal fashion. From the early maturation phase through maturation of the spermatids the fibers of the nuceloli appeared to be replaced by a fine filamentous structure. As the condensation of chromatin proceeded, the filamentous structure decreased in density, and finally the space of the nucleolus was replaced by a nucleolar vacuole.

Neck region of gerbil spermatozoa

AbstractIn the course of the reorganization and degeneration of the proximal centriole in the mature acentriolate spermatozoon of the Mongolian gerbil, both the proximal and distal centrioles appear in the early cap phase of spermatid development. During the acrosome phase, both distal and proximal centrioles become highly active in the formation of a segmented column. The proximal centriole becomes actively involved in the formation of the capitulum, while the distal centriole forms the axonemal complex and dense fibers. During the maturation phase of spermatid development, the “pinwheel” arrangement of the proximal centriole becomes an “S”‐shaped structure, turned 90° on its vertical axis. The few “doublet” microtubules that can be detected later in that stage completely disappear during spermiation. The distal centriolar area develops a single central pair of microtubules and membranous elements. Another prominent feature in the neck region of the gerbil spermatozoa is the presence of two dense rudimentary columns in association with the mitochondria. Although their density is similar to that of the other columns, these two columns have no connection with the dense fibers; in fact, they are closely associated with the mitochondria.

A “Living Cell Method” for Testing the Early Effects of Antispermatogenic Compounds: Model Experiments with Two Alkylating Agents ThioTepa and Nitrogen Mustard

An accurate identification of stages of spermatogenesis in freshly isolated unstained seminiferous tubules is possible by a simple transillumination. Physiologically, the acrosome and maturation phase spermatids are arranged in bundles or as a continous layer and give rise to light absorption patterns characteristic for different cell associations. The damaged and dying cells cause alterations in the light absorption pattern which helps their specific selection for further analyses by morphological and biochemical methods.In model experiments, we have analyzed the early stage‐specific effects of two alkylating anticancer agents, nitrogen mustard and ThioTepa. Increased light absorption was seen in segments V–VI caused by degenerating early spermatids and mid‐pachytene spermatocytes, and in segments XII–I caused mainly by pachytene spermatocytes. Phase contrast microscopy proved to be valuable in analysis of the phagocytosing function of the Sertoli cells. Electron microscopy revealed that probably the earliest observable alterations were disintegrations of the developing acrosomic system, of the chromatoid body in early spermatids and of the spermatid nuclei during the late acrosome phase. Biochemical analyses revealed an inhibition of physiological DNA and RNA syntheses and an activation of unscheduled repair‐DNA synthesis.The “living cell method” gives new possibilities for combined morphological and biochemical analyses of specific effects of antifertility and anticancer drugs on different stages of mitotic and meiotic cell cycles during spermatogenesis.

Evolution of the endoplasmic reticulum during rat spermiogenesis.

AbstractThe endoplasmic reticulum (ER) was stained selectively by a sequence of uranyl acetate, lead and copper citrate, according to the method of Thiéry and Rambourg ('76), and then investigated in 0.5 to 2.0‐μm‐thick sections with the transmission electron microscope. Examination of photographic stereopairs allowed a three‐dimensional visualization of the ER at various steps of spermiogenesis.During the Golgi and cap phases of spermiogenesis (steps 1–7, classification of Leblond and Clermont, '52a), the ER is distributed throughout the cytoplasm as a three‐dimensional network of spherical and tubular cisternae connected by narrow tubules. In addition, a close network of tubular cisternae is located along the convex surface of the Golgi apparatus and lines the plasma membrane. Where the cell membrane joins that of another spermatid to form an intercellular bridge, this network extends across the bridge.During the acrosome phase (steps 8–14), the cytoplasm contains an abundant ER that shows the following modifications: (A) Along the inside and outside of the caudal tube the cisternae form long tubes or plates which run adjacent and parallel to the microtubules. These cisternae are connected by delicate lateral anastomoses; (B) Along the flagellum the ER forms a “fenestrated sleeve” made up of a close network of tubular cisternae; (C) Similar networks are organized as “fenestrated spherules” enclosing large vesicles seen throughout the cytoplasm; (D) At a short distance from the flagellum, the ER cisternae are continuous with a stack of annulate lamellae and an aggregate of radially arranged collapsed cisternae called the “radial body”.During the last or maturation phase (steps 15–19), the ER regresses. Thus, during the final steps in the formation of the flagellum, the ER network fragments and then most of the cisternae disappear from the cytoplasm. The “radial body” is the last element of the ER to be dissolved. Thus the ER undergoes extensive structural modifications during spermiogenesis, suggesting an active contribution of this organelle to the differentiation of the spermatid into a spermatozoon.

Relative volume of sertoli cells in monkey seminiferous epithelium: A stereological analysis

Techniques of quantitative stereology have been utilized to determine the relative volume occupied by the Sertoli cells and germ cells in two particular stages (I and VII) of the cycle of the seminiferous epithelium. Sertoli cell volume ranged from 24% in stage I of the cycle to 32% in stage VII. Early germ cells occupied 3.4% in stage I (spermatogonia) and 8.7% in stage VII (spermatogonia and preleptotene spermatocytes). Pachytene spermatocytes occupied 15% (Stage I) and 24% (stage VII) of the total volume of the seminiferous epithelium. In stage I the two generations of spermatids comprised 58% of the total epithelium by volume, whereas in stage VII, after spermiation, the acrosome phase spermatids occupied 35% of the total seminiferous epithelial volume.

The influence of progestin and androgen on the fine structure of the male reproductive tract of the rat. I. General effects and observations on the testis.

The combination of a progestin and androgen has received attention as a possible male contraceptive. The progestin is thought to reduce gonadotropin release and suppress spermatogenesis, while the sex accessory organs and male characteristics are maintained by the simultaneous administration of testosterone. In the present study, the histology and ultrastructure of parts of the male reproductive tract of rats treated with medroxyprogesterone (Provera, Upjohn) (1 mg/100 g body weight/day) alone and combined with testosterone (15, 30, or 100 mug/100 g/day) were studied following treatment for up to 16 weeks. The testes and epididymides of rats administered Provera alone or Provera and testosterone weighed less than those of control rats. The weights of the accessory glands of rats treated with Provera were greatly reduced; it was possible to maintain them at approximately control levels by simultaneously administering sufficient testosterone (100 mug/100 g body weight/day). The fertility of some of the animals was tested by caging them with female rats, and none of the treated rats tested in this way was fertile. Similar microscopic alterations were present in the testes of animals administered Provera alone or Provera and different levels of testosterone. Spermatogonia, spermatocytes, and early spermatids were abundant in treated rats and did not show ultrastructural changes. However, many degenerating or necrotic spermatids of the cap phase (approximately stages 6-7) and later were present. Late spermatids of the acrosome and maturation phases were rare. Some necrotic spermatids were surrounded by Sertoli cells, and parts of spermatids lay within lysosome-lyke structures in the cytoplasm of Sertoli cells. Many large lipid droplets were also present in Sertoli cells of treated rats. Leydig cells were smaller in treated animals than in control rats. The results suggest that germ cells can develop up to cap phase spermatids but then undergo degeneration. These alterations in spermatogenesis may be responsible in large part for the antifertility effect of the progestin and androgen combination. Some rats were permitted to recover following the end of treatment. The microscopic appearance of the testis returned to normal within three to six weeks, although epididymal alterations persisted in some animals six weeks after the end of treatment. By 9 to 12 weeks after the end of treatment the reproductive organs had a normal microscopic appearance in all the rats studied.

A scheme of rabbit spermateleosis based upon electron microscopical observations

Rabbit spermateleosis is subdivided into ten stages (A to J) based upon fine structure. Spherical spermatids in stages A to D correspond to the Golgi phase and the cap phase of Leblond and Clermont (1952a). Elongating spermatids in stage E through the first half of stage H correspond to the acrosome phase, and maturating spermatids in the second half of stage H through stage J correspond to the maturation phase. The development of the different organelles during each stage is described and put into a scheme of normal rabbit spermateleosis that can be used as a reference in experimental investigations of ultrastructural changes.

CYCLICAL CHANGES IN AND SIGNIFICANCE OF LIPIDS AND ACID PHOSPHATASE ACTIVITY IN THE SEMINIFEROUS TUBULES OF THE RAT TESTIS.

AbstractThe distribution of tubular lipids and acid phosphatase activity was studied in the rat testis and in the ductuli efferentes. The results were referred to the stage of the cycle of the seminiferous epithelium. The significance of the lipids and phosphatase of the ducts was tested in animals, where excretory ducts were separated from testis for 30 to 40 days.During the acrosome phase of the old generation of spermatids the lipids first made their appearance in the cytoplasm. During the subsequent stages of the cycle (II to VIII) both the amount of lipids and the intensity of the acid phosphatase activity gradually increased, reaching a maximum in the cytoplasmic “split‐offs” of the mature sperm at stage VIII. Most of the lipids, together with phosphatase‐positive cytoplasmic granules were discarded with the spermatozoa, but a small proportion underwent phagocytosis by the Sertoli cells. During stage IX both lipid‐ and phosphatase‐positive particles moved to the periphery of the tubules as their number in the basal cytoplasm of the Sertoli cells rapidly increased. Through the stages IX to II a gradually diminishing amount of lipids and acid phosphatase activity was seen forming a peripheral ring in the tubules. A temporary rise in the Sertoli cell phosphatase activity took place at the time when the nuclei of the maturing spermatids were deep in the seminiferous epithelium (stages IV and V).Lipid droplets associated with acid phosphatase activity were present in the epithelium of ductuli efferentes of intact rats. Ductectomy caused a complete disappearance of the epithelial lipids. Simultaneously, cysts filled with degenerating spermatozoa and abundant lipids formed on the cut surface of the testis. It is concluded that the main part of the residual cytoplasm of the spermatids forms an apocrine secretion which is presumably absorbed in the ductuli efferentes and in other parts of the excretory duct system.

EFFECT OF OVINE GONADOTROPHINS AND ANTISERUM TO INTERSTITIAL CELL-STIMULATING HORMONE ON THE TESTIS OF THE HYPOPHYSECTOMIZED RAT.

ABSTRACT Rabbit antiserum to ovine ICSH was administered to male Sprague-Dawley rats 2 months after hypophysectomy; further atrophy of the testis ensued. The interstitial tissue, the Sertoli cells, and the germ cells were adversely affected by the treatment. In the absence of gonadotrophins and exogenous androgen, spermatogenesis was nearly arrested. Ovine ICSH repaired the interstitial tissue and stimulated mitotic activity among the spermatogonia. Ovine FSH (NIH-FSH-S-1), containing approximately 1% ICSH activity, stimulated spermatid formation. When antiserum to ICSH was injected together with the FSH, no maturation divisions or spermatids were observed. The combination of FSH and ICSH, 100 μg of each per day injected for a period of 21 days, stimulated extensive and uniform tubular repair and advanced spermiogenesis to the acrosome phase; an enhancement in the secretion of androgen also was effected when FSH was given in combination with ICSH.

The induction of dominant lethal mutations in rats by alkane sulphonic esters

Sterility resulting from treatment of male rats with methyl ethane sulphonate and ethyl methane sulphonate was found to be due to the induction of dominant lethal mutations in spermatozoa and spermatids. Spermatids in the acrosome phase of development were found to be the most sensitive cells and at low doses of both compounds the majority of deaths occurred after implantation. The earlier cell stages, i.e. spermatogenia and spermatocytes, were the most sensitive to isopropyl methane sulphonate and Busulphan (Myleran). The action on spermatogonia was confirmed by spermatid counts and the time of occurrence of oligospermia or aspermia was calculated. Pre-implantation deaths occurred mainly in the early cleavage stages after both isopropyl methane sulphonate and Busulphan; at least part of these losses can be attributed to the induction of dominant lethal mutations in spermatocytes and early spermatids. Isopropyl methane sulphonate appears to be the more effective of the two drugs in this respect.

Reinitiation and Restoration of Spermatogenesis with Testosterone Propionate and Other Hormones After a Long-Term Post-hypophysectomy Regression Period

The ability of testosterone propionate (TP) to restore spermatogenesis was investigated in 67- to 70-day hypophysectomized rats administered TP either separately or in combination with somatotrophin (STH) and/or thyroxine (T4). Following TP treatment for 35 days, the testicular weights had increased to twice those of the hypophysectomized controls, and a few of the seminiferous tubules contained germinal cells as mature as acrosome phase spermatids. With more prolonged therapy (90–110 days), spermatogenesis was restored qualitatively but not quantitatively in 33% of the animals, while the remainder of the animals showed varying degrees of response. The testes of animals given TP+STH or TP+STH+T4 for 35 days were heavier than those of animals receiving TP alone, and a few of the seminiferous tubules contained maturation phase spermatids. Treatment for 90–110 days completely restored spermatogenesis in ½ of these animals. Consequently, although testosterone alone can restore spermatogenesis to a limited extent, the addition of STH appears to augment this effect significantly. Following total post-hypophysectomy regression of the testes, some seminiferous tubules apparently lose the ability to respond to hormonal stimulation. In contrast, complete morphologic and functional restoration of the male reproductive system is possible in some animals if therapy is continued for sufficient periods of time.

Role of the parathyroids in the production of osteitis fibrosa and soft tissue calcification by lead acetate.

The ability of testosterone propionate (TP) to restore spermatogenesis was investigated in 67- to 70-day hypophysectomized rats administered TP either separately or in combination with somatotrophin (STH) and/or thyroxine (T4). Following TP treatment for 35 days, the testicular weights had increased to twice those of the hypophysectomized controls, and a few of the seminiferous tubules contained germinal cells as mature as acrosome phase spermatids. With more prolonged therapy (90–110 days), spermatogenesis was restored qualitatively but not quantitatively in 33% of the animals, while the remainder of the animals showed varying degrees of response. The testes of animals given TP+STH or TP+STH+T4 for 35 days were heavier than those of animals receiving TP alone, and a few of the seminiferous tubules contained maturation phase spermatids. Treatment for 90–110 days completely restored spermatogenesis in ½ of these animals. Consequently, although testosterone alone can restore spermatogenesis to a limited ext...

Histochemical evidence for cross-linking of DNA by alkylating agents in vivo

Unfixed, frozen sections of rat liver, spleen, thymus and testis were extracted with strong salt solution. This treatment removed nearly all the DNA from liver, spleen and thymus and, in the testis, from Sertoli cells, spermatogonia, spermatocytes and early spermatids. Spermatid DNA became increasingly inextractable after the end of the acrosome phase and completely so by the end of the maturation phase, when spermatozoa were formed. In animals treated with triethylene melamine or nitrogen mustard a further, marked loss of extractability was superimposed on the normal changes, beginning within a few minutes of intraperitoneal injection and most evident in spermatids at the late acrosome and early and mid-maturation phases. Monofunctional alkylating agents did not have this effect. Triethylene melamine did not induce resistance to extraction in the DNA of liver, spleen or thymus. The naturally occurring spermatid changes appear to be due to the development of an insoluble protein sheath, and the superimposed loss of solubility induced by alkylating agents may be attributed to the direct or indirect cross-linking of DNA to this.