Sperm nuclear vacuoles in relation to acrosome reactions and sperm motility.

We investigated sperm nuclear vacuolation in relation to acrosome reactions and the maintenance of sperm motility. Thirty male patients who visited our Male Infertility Clinic were enrolled. These patients underwent conventional semen analyses, Acrobeads tests, and high-magnification observation of the sperm head to evaluate the degree of nuclear vacuolation on the Acrobeads test scoring after 24 hours of incubation. The presence of acrosome reactions was evaluated using the Acrobeads test. The spermatozoa were classified into three groups: (I) those bound to MH61-beads, (II) motile spermatozoa that did not bind to MH61-beads, and (III) immotile spermatozoa that did not bind to MH61-beads. The percentage of spermatozoa with large nuclear vacuoles (%LNV) was compared between the three groups. The degree of sperm nuclear vacuolation was evaluated in 17,992 ejaculated spermatozoa. The mean %LNVs were 2.4% in group I, 5.8% in group II, and 9.8% in group III. These values were significantly different from each other (P < 0.001, paired t-test). There were no correlations between the %LNV values and the Acrobeads scores. In conclusion, the degree of sperm nuclear vacuolation was significantly lower in the acrosome-reacted spermatozoa and spermatozoa with maintained motility, and higher in the immotile spermatozoa that did not bind to MH61-beads.

PACAP-mediated sperm-cumulus cell interaction promotes fertilization.

The developing acrosome in spermatids contains pituitary adenylate cyclase-activating polypeptide (PACAP). However, the role of the acrosomal PACAP remains unclear because it has not been detected in mature spermatids and sperm. We reinvestigated whether the sperm acrosome contains PACAP. An antiserum produced against PACAP reacted to the anterior acrosome in epididymal sperm fixed under mild conditions, suggesting that PACAP acts on oocytes and/or cumulus cells at the site of fertilization. Immunolabeling and RT-PCR demonstrated the presence of PACAP type I receptor, a PACAP-specific receptor, in postovulatory cumulus cells. To investigate the role of PACAP in fertilization, we pretreated cumulus-oocyte complexes with the polypeptide. At a low concentration of sperm, the fertilization rate was significantly enhanced by PACAP in a dose-dependent manner. Sperm penetration through the oocyte investment, cumulus layer, and zona pellucida was also enhanced by PACAP. The enhancement was probably due to an enhancement in sperm motility and the zona-induced acrosome reaction, which were stimulated by a cumulus cell-releasing factor. Indeed, PACAP treatment increased the secretion of progesterone from the cumulus-oocyte complexes. These results strongly suggest that in response to PACAP, cumulus cells release a soluble factor that probably stimulates sperm motility and the acrosome reaction, thereby promoting fertilization.

Autophagy is activated for cell survival after endoplasmic reticulum stress.

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

Nerve fiber analysis on the so-called accessory subscapularis muscle and its morphological significance.

A rare muscular anomaly, so-called accessory subscapularis muscle, was found in the left axillary fossa of a 95-year-old male cadaver during a student dissection practise. The muscle arose near the lateral margin of the scapula from the surface of the subscapularis muscle and ran upward to fuse with the capsule of the shoulder joint via a tendon. It measured 1.0 cm in width, 7.0 cm in length and 1.5 mm in thickness, and was separated from the underlying subscapularis muscle by the axillary and inferior subscapular nerves. Macroscopically, the anomalous muscle received its nerve supply from a branch arising from the lower root of the radial nerve near the origin of the thoracodorsal nerve and entered the muscle from its ventral surface. Nerve fiber analysis showed that the supplying nerve originated from fibers of the dorsal element of C7 immediately cranial to the thoracodorsal nerve. These findings indicate that the present anomalous muscle might be close to the formation of the latissimus dorsi muscle in its derivation rather than the subscapularis muscle.

Superficial brachial artery crossing over the ulnar and median nerves from posterior to anterior: embryological significance.

A rare variation of the superficial brachial artery was found in the right arm of an 82-year-old male cadaver in student dissection practice. In this case, the axillary artery passed normally between the medial and lateral roots of the median nerve and then bifurcated into a large superficial brachial artery and the deep brachial artery (A. brachialis profunda). The superficial brachial artery passed medially to the ulnar nerve and then crossed over to take a lateral course to the median nerve at the midpoint of the upper arm. It finally divided into the radial and ulnar arteries at the cubital fossa. The deep brachial artery terminated as the inferior ulnar collateral artery. These findings indicate that the present variant was a well-developed medial type of the superficial brachial artery that gave off the ulnar and radial arteries. The anatomical and embryological significance of the findings are discussed.

Morphological study of a horseshoe kidney with special reference to the vascular system.

This is a morphological study on an autopsy case of horseshoe kidney found in a 79-year-old-female cadaver. This kidney consisted of two distinct renal masses that were connected at their lower poles by a parenchymal isthmus that was located in the front of the abdominal aorta at the level of the fourth lumbar vertebra. The kidney was supplied by four arteries arising from the abdominal aorta. The distribution of intrarenal arteries showed that the nature of segmental arteries in the present case was basically the same as in the normal kidney, except that the isthmus had its own blood supply from the artery directly arising from the aorta approximately 28 mm below the origin of the inferior mesenteric artery. Venous drainage from the kidney, including the isthmus, was taken by three veins that opened independently into the inferior vena cava. No congenital malformations were found in other organs. We discuss the anatomical and embryological significance of this anomaly and its associated vascular system.

An aberrant axillary artery penetrating the origin of the radial nerve from deep to superficial.

An aberrant axillary artery that penetrated the radial nerve from deep to superficial during its course, was observed. The brachial plexus in the present case was classified as the Adachi's C-type brachial plexus. Further, an accessory radial root existed, which was a nerve bundle branching from the deep aspect of the inferior trunk and communicating with the radial root from the posterior cord to form the radial nerve. The axillary artery went on along the lower border of the brachial plexus and passed between the radial root and the accessory radial root from deep to superficial at its third section. As the axillary artery penetrated the origin of the radial nerve from deep to superficial, it was judged to reach deep under the posterior cord, hence deeper than the brachial plexus.

Changes in the activity of sperm nitric oxide synthase in the oviductal reservoir during ovulation.

Background: The oviductal isthmus is known to act as a sperm reservoir in several mammalian species including mice, but it is still unclear how sperm are released from the reservoir after ovulation. Recently, nitric oxide (NO) was reported to have important roles as a mediator in various sperm functions, including hyperactivation and capacitation. Therefore, we have investigated the change of the activity of nitric oxide synthase (NOS) of sperm of the isthmus in relation to ovulation under in vivo fertilization conditions. Methods and Results: The sperm were collected from the isthmus and uterus of the female mated before or after ovulation. The NOS activity change was evaluated by using the ß-nicotinamide adenine dinucleotide phosphate-diaphorase staining method, and sperm NOS activity was quantified by using NIH image software. The results showed that, in the reservoir, the peak intensity of sperm NOS activity was higher after ovulation (135.5 ± 22.4) than before ovulation (102.7 ± 15.5; P ≤ 0.05). After ovulation, the number of free sperm in the isthmus increased, and these sperm expressed strong NOS activity. Conclusion: The change of sperm NOS activity is related to their release from the epithelium of the oviductal reservoir. (Reprod Med Biol 2003; 2: 75-81).

Preferential localization of rat GAPDS on the ribs of fibrous sheath of sperm flagellum and its expression during flagellar formation.

The proper assembly of sperm flagellar proteins is fundamental for sperm motility. The sperm- and spermatid-specific isoform of glyceraldehyde 3-phosphate dehydrogenase, GAPDS, is a flagellar protein indispensable for sperm flagellar movement. To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody. Immunolocalization of rat GAPDS was restricted to the fibrous sheath (FS) in the sperm flagellum, and was predominant in the circumferential ribs rather than the longitudinal columns. Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation. Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

Change in expression of a 90-kDa glycoprotein GP90-MC301 during prenatal and postnatal testicular development: a differentiation marker for rat germ cells.

GP90-MC301, a 90-kDa glycoprotein recognized by the monoclonal antibody MC301, is a reliable stage-specific marker for preleptotene to pachytene spermatocytes in adult rat testes. In this study we confirmed that the glycoprotein is also useful as a marker for germ cells in prenatal and postnatal testes. Immunohistochemical analysis showed a dramatic change in GP90-MC301 expression in germ cells during testis development. Strong expression was detected in primordial germ cells at embryonic day (E) 13 and in gonocytes at E16, and the expression was then markedly reduced at around the time (E18) gonocytes undergo G1/G0 arrest, and was not restored in gonocytes or spermatogonia afterward. Thereafter, it reappeared in primary spermatocytes in the prepubertal period. Testicular somatic cells such as Sertoli cells, Leydig cells, and peritubular myoid cells expressed GP90-MC301 during specific periods which were largely correlated with periods of active proliferation of these testicular somatic cells. Western blotting showed that GP90-MC301 was expressed during testis development without a change in its molecular size. Thus, GP90-MC301 is potentially useful for the analysis of not only spermatogenesis but also early testis development.