Functional anatomy of the male reproductive system of the American lobster (Homarus americanus).

Despite supporting a valuable fishery, the reproductive system of the male American lobster (Homarus americanus) is poorly understood. The elongated H-shaped testis is responsible for spermatogenesis and is composed of follicles, a common collecting duct with interlaced scattered striated muscles, and a serosa as an external wall. Sertoli cells are associated with the spermatogenesis that produces spermatozoa, which are transferred to the collecting duct through a temporary passageway. Spermatogenesis is asynchronous between follicles and occurs on a continuous basis. The anterior and posterior lobes of the testes are independent and connect to the vasa deferentia through the Y-shaped collecting tubules that have a different cell anatomy and function than the two organs they connect. The vas deferens is divided into four regions. Spermatophores, produced in the proximal vas deferens, are packets of spermatozoa encapsulated in a single layer-the spermatophoric wall, which is composed of mucopolysaccharide acid. Large dense ovoid granules and the seminal fluid, composed of acidic sulfated mucosubstances, are secreted in the median vas deferens. Spermatophores within these secreted substances (i.e., semen) are stored in the distal vas deferens that, with the spermiduct (last region of the vas deferens), is responsible for the extrusion of the semen by striated muscle contractions. Smooth muscles suggest a peristaltic movement of the spermatophores within the vas deferens. Finally, the gonopores and the first pair of pleopods (i.e., gonopod) move the semen to the female seminal receptacle during copulation.

Vitamin B12-induced spermatogenesis recovery in cimetidine-treated rats: effect on the spermatogonia number and sperm concentration.

The H2-receptor antagonist cimetidine is an antiulcer drug also used for the treatment of cancer due to its antiangiogenic effect. However, this drug has caused structural changes in the seminiferous tubules. Vitamin B12 has been used as a therapeutic agent for the treatment of male infertility. The supplementation of rats with vitamin B12 during cimetidine treatment has recovered the damaged seminiferous tubules, but how this vitamin restores the seminiferous epithelium has not been clarified. In this study, we evaluated whether vitamin B12 improves the number of spermatogonia, spermatocytes, and sperm concentration in cimetidine-treated rats. Adult male rats were treated for 50 days as follows: cimetidine group received 100 mg kg-1 b.w. of cimetidine, cimetidine-B12 group received cimetidine and 3 µg of vitamin B12-hydroxocobalamin, B12 group received only 3 µg of vitamin, and control group received saline. Sperm concentration was calculated and historesin-embedded testes sections were used for the quantitative analyses of spermatogonia (A; In/B) and spermatocytes. TUNEL method and PCNA immunofluorescence were performed. Cimetidine caused a significant reduction in sperm concentration. TUNEL-positive spermatogonia and spermatocytes were correlated to a significant reduction in the number of these cells. In cimetidine-B12 group, sperm concentration was higher than cimetidine group and a significant increase in the number of spermatogonia (stages II-VI) was correlated to a high incidence of PCNA-immunolabeled spermatogonia and spermatocytes. The results show that the supplementation of rats with vitamin B12 during cimetidine treatment increases sperm concentration and exerts a potential effect in the recovery of spermatogonia and spermatocytes.

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.

Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro.

The genetic manipulation of spermatogonial stem cells (SSCs) can be used for the production of transgenic animals in a wide range of species. However, this technology is limited by the absence of an ideal culture system in which SSCs can be maintained and proliferated, especially in domestic animals like the goat. The aim of this study therefore was to investigate whether the addition of vitamin C (Vc) in cell culture influences the growth of caprine SSCs. Various concentrations of Vc (0, 5, 10, 25, 40, and 50 µg/mL(-1)) were added to SSC culture media, and their effect on morphology and alkaline phosphatase activity was studied. The number of caprine SSC colonies and area covered by them were measured at 10 days of culture. The expression of various germ cell and somatic cell markers such as VASA, integrins, Oct-4, GATA-4, α-SMA, vimentin, and Thy-1 was studied to identify the proliferated cells using immunostaining analyses. Further, the intracellular reactive oxygen species (ROS) level was measured at the 3rd, 6th, and 9th day after culture, and expression of Bax, Bcl-2, and P53, factors involved in the regulation of apoptosis, were analyzed on the 7th day after culture using reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The results showed that the SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphologic differences between the groups. The number and area of colonies were both the highest in the 40 µg/mL(-1) Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia, and testis somatic cells was observed. Cultured germ cell clumps were found to have alkaline phosphatase activity regardless of the Vc dose. The number of Thy-1- and Oct-4-positive cells was the most in the 40 µg/mL(-1) Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 µg/mL(-1) was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptotic genes Bax and P53. In conclusion, the addition of 40 µg/mL(-1) Vc can maintain a certain physiological level of ROS, trigger the expression of the antiapoptosis gene Bcl-2, suppress the proapoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro.

Internal anatomy and ultrastructure of the male reproductive system of the Norway lobster Nephrops norvegicus (Decapoda: Astacidea).

The Norway lobster (Nephrops norvegicus) is economically important in Europe. However, apart from the female reproductive system, very little is known about its internal anatomy. This article focuses on studying the internal anatomy and ultrastructure of the male reproductive system. This system follows the general pattern found among decapod crustaceans, with several peculiarities. Testes are composed of lobular sperm ducts in which the spermatozoa are fully constituted. The spermatozoa present three lateral arms and a long acrosome, which gives a false appearance of flagellated spermatozoa. The two testes form a double H under the heart, and the vas deferens (VD) arise from each side at the posterior edge of the double H. The main characteristic of the VD is the presence of a sphincter in the enlarged area of the distal end of the middle VD. The MVD here shows an increase in musculature of the wall as compared to the VD, which regulates the passage of the sperm cord to the distal VD (DVD) and thence to the thelycum of the female. The wall of the spermatophore is formed in the distal part of the proximal VD, which surrounds the unique sperm cord present in the VD. Isolated spermatophores are not observed in the VD. The sperm cord is pinched off during copulation by the musculature of the DVD. Then, a portion of the sperm cord is transferred from each VD to form the isolated spermatophores. The wall of the spematophores and the spermatozoa that are observed inside the thelycum have the same morphology as those observed in the VD.

Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium.

Cytogenetic damage induced by a wide range of concentrations of uranyl fluoride injected into mouse testes was evaluated by determining the frequencies of chromosomal aberrations in spermatogonia and primary spermatocytes. Breaks, gaps and polyploids were observed in spermatogonia. The frequencies of the significant type of aberration, breaks, were induced according to the injected doses of uranyl fluoride. Primary spermatocytes were examined for fragments, univalents and multivalents. The multivalents observed in this study resulted either from chromatid interchanges or from reciprocal translocations. The reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes. For primary spermatocytes the incidence of aberrant cells largely depended on the administered dose. Sampling time after treatment could affect the frequencies of chromosomal aberrations in male mouse germ cells.

An abbreviated method of the double lead stain technique.

An enhanced staining of cellular organelles and cytoplasmic matrix is observed when ultrathin sections for electron microscopy are stained for 1-5 min in fresh Reynolds' lead citrate, rinsed in distilled water and dried prior to staining with a saturated solution of uranyl acetate (40 min) and Reynolds' lead citrate (20 min), Daddow (1983). The above procedure was recommended for tissues with poor staining qualities either from prolonged fixation or inadequacies of the buffer or the embedding medium used. Using an abbreviated method of the above (i.e., 30 sec lead citrate, 60 sec saturated uranyl acetate and 30 sec lead citrate), overfixed or normally fixed tissues show enhanced contrast. Sodium hydroxide (0.02 M), substituted for the initial lead stain, increases contrast to a lesser extent.