Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii.

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

Histological development of human testicular cords from 70 to 90 days of gestation.

The development of the testicular cord structure was investigated in 4 human fetuses between 70 and 90 days of gestation, in which the testicular cords are differentiating into the seminiferous tubules. Histological examinations were performed using stains with haematoxylin-eosin (HE), Masson's trichrome (MT), periodic acid schiff (PAS), anti-proliferating cell nuclear antigen (PCNA) monoclonal antibodies, and TUNEL methods. It was found that the testicular cords structures were indefinitely observed in HE-stained sections of four fetuses. However, the basement membranes of the testicular cord were clearly stained with MT, showing the tubular structure. Furthermore, cells in the testicular cords were positive with PAS, but the interstitial tissues outside the testicular cords were negative. PCNA-positive cells were detected not only inside but also outside the testicular cords, however, TUNEL positive cells are not detected throughout all testicular tissues.

[Effects of diethylstilbestrol on cultured testis gubernacular cells in mice].

OBJECTIVE: To establish a primary culture of the testis gubernacular cells of Kunming mice, observe the morphological characteristics of the cells, and explore the effects of exogenous estrogens (EEs) on the development of the testis gubernacula in vitro. METHODS: We removed the gubernacula from 3-day-old mice with the surgical magnifier and cultured the gubernacular cells. Then we detected the cell viability by trypan blue and cell morphology by HE staining. The subcultured cells were randomly divided into a blank control, a DMSO (0.1%, v/v) control, and 4 experimental groups (given 0.01, 0.10, 1.00 and 10.00 micdrog/ml of diethylstilbestrol [DES] dissolved in DMSO, respectively). After treated for 12, 24 and 48 hours, the gubernacular cells were observed for morphological changes and proliferation inhibition by CCK-8. RESULTS: Most of the cultured gubernacular cells were fibroblasts, and a few were epithelioids. The primary cells showed a viability of 85%-90%. Dose- and time-dependent inhibition of cell proliferation was found in the four experimental groups at three different times, with statistically significant differences (P < 0.01). CONCLUSION: Gubernacular cells can be cultured in vitro. EEs inhibit the proliferation of gubernacular cells in a dose- and time-dependent manner. An in- sight into the effects EES on cultured gubernacular cells is an effective approach to the study of their influence on the development of the reproductive system.

A critical time window of Sry action in gonadal sex determination in mice.

In mammals, the Y-linked sex-determining gene Sry cell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9), leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However, how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns.

Peritubular myoid cells are not the migrating population required for testis cord formation in the XY gonad.

Cell migration is one of the earliest events required for development of the testis. Migration occurs only in XY gonads downstream of Sry expression and is required for the subsequent epithelialization of testis cords. Using organ culture experiments and tissue recombination, we and others speculated that peritubular myoid (PTM) cells were among the migratory cells and were likely the cell type required for cord formation. However, because no unique marker was found for PTM cells, their positive identification during or after migration remained unclear. alpha-Smooth Muscle Actin (alphaSma; approved gene symbol Acta2), a classic marker of adult PTM cells,is expressed broadly in testis interstitial cells at E12.5, and becomes highly enriched in PTM cells by E15.5-16.5. We used a novel transgenic line expressingEYFP under the control of an alphaSma promoter to determine whether alphaSma-EYFP positive cellsmigrate into the gonad. Surprisingly, mesonephroi expressing alphaSma-EYFP do not contribute any EYFP positive cells to XY gonads when used as donors in recombination cultures. These results indicate that alphaSma-EYFP cells do not migrate into the gonad during the critical window of sex determination and cannot be the migrating cell type required for testis cord formation. Our results suggest that PTM cells, and most other interstitial lineages, with the exception of endothelial cells, are induced within the gonad. These experiments suggest that endothelial cells are the migrating cell type required for epithelialization of testis cords.

A new method of culturing Leydig cells and prospects of their practical use in andrology.

We present a new method of obtaining a xenoculture enriched with Leydig cells from neonatal porcine testes providing 97-99% viability of cells in the culture. Genital extra-testicular transplantation of the obtained xenoculture was applied, which minimized traumaticity of surgery and the number of postoperative complications and had undoubted advantages over the subcapsular cell culture depot.

Mitotic activity of Sertoli cells in adult human testis: an immunohistochemical study to characterize Sertoli cells in testicular cords from patients showing testicular dysgenesis syndrome.

During puberty, normal somatic Sertoli cells undergo dramatic morphological changes due to the differentiation of immature pre-Sertoli cells in functionally active adult Sertoli cells. Sertoli cell maturation is accompanied with loss of their mitotic activity before onset of spermatogenesis and loss of pre-pubertal and occurrence of adult immunohistochemical Sertoli cell differentiation markers. Testes of infertile adult patients often exhibit numerous histological signs of testicular dysgenesis syndrome (TDS) such as microliths, Sertoli cell only (SCO) tubules, tubules containing carcinoma in situ and immature seminiferous tubules (Sertoli cell nodules). Sertoli cell tumours, however, are very rare neoplasms possibly due to the fact that the mechanism and temporal origin of neoplastic Sertoli cells underlying Sertoli cell tumourigenesis still remain unknown. To clarify the state of Sertoli cell differentiation in both immature seminiferous tubules of adult patients with TDS and Sertoli cell tumour, we compared the expression of the Sertoli cell differentiation markers vimentin, inhibin-alpha, anti-Muellerian-hormone, cytokeratin 18, M2A-antigen, androgen receptor and connexin43 with that of SCO tubules with hyperplasia. In addition, we demonstrated for the first time the existence of proliferating Sertoli cells by Ki67- and PCNA-immunostaining in Sertoli cell nodules of the adult human testis. Our data indicate that mitotically active Sertoli cells in Sertoli cell nodules will be arrested prior to puberty and, contrary to dogma, do not represent foetal or neonatal cells. Since all markers in Sertoli cell nodules revealed a staining pattern identical to that in neoplastic Sertoli cells, but different to that in Sertoli cells of SCO tubules with hyperplasia, it may be speculated that Sertoli cell tumours in adult men may originate from Sertoli cell nodules.

Influence of tolrestat on the defective leukocyte-endothelial interaction in experimental diabetes.

One of the most devastating secondary complications of diabetes is the blunted inflammatory response that becomes evident even in the very early stages of poorly controlled diabetes mellitus. While the etiology of this diminished response is not clearly understood, it has been linked to a decrease in the respiratory burst of neutrophils, as well as a decrease in microvessel response to inflammatory mediators and defective leukocyte-endothelial interactions. Using video microscopy to visualize vessels of the internal spermatic fascia, we have characterized leukocyte-endothelial interactions in alloxan-induced diabetic and in galactosemic rats by quantitating the number of leukocytes rolling along the venular endothelium and the number of leukocytes sticking to the vascular wall after topical application of zymosan-activated plasma or leukotriene B(4) (1 ng/ml), as well as after the application of a local irritant stimulus (carrageenan, 100 microg). We observed that while 33 days of alloxan-induced diabetes or 7 days of galactosemia had no effect on total or differential leukocyte counts and on the wall shear rate, both treatments significantly (P<0.001) reduced the number of leukocytes rolling along the venular endothelium by about 70% and the number of adhered leukocytes in postcapillary venules by 60%. These effects were not observed in diabetic and galactosemic animals treated with an aldose reductase inhibitor. The results suggest that impaired leukocyte-endothelial cell interactions are a consequence of an enhanced flux through the polyol pathway.

Morphological features of the spermatic cord in the musk shrew (Suncus murinus) with special reference to extratesticular Leydig cells.

Morphological features of the testicular artery and vein in the spermatic cord of the musk shrew (Suncus murinus) were evaluated by light microscopy, transmission electron microscopy, corrosion cast technique combined with scanning electron microscopy and immunohistochemistry. The vascular architecture in the spermatic cord of the musk shrew was simple. The testicular artery in the musk shrew was straight and accompanied by 1 to 3 branches of testicular vein. The testicular vein was also straight and anastomosed with each other in some points along its length, but it did not form a delicate pampiniform plexus. In the middle and distal portions of the spermatic cord, the tunica adventitia of the artery and vein was joined together to form a single connective tissue septum. Clusters of cells were found in this connective tissue septum in the middle portion of the cord. These cells were located close to the arterial wall and nerve endings, but they did not appear inside of neurium. They showed several typical characteristics similar to Leydig cells, and they were positive for 3beta hydroxysteroid dehydrogenase (HSD) antibody. Ultrastructural and immunohistochemical studies also indicated that the cells in cluster found in the vascular wall of the musk shrew spermatic cord may be equivalent to Leydig cells in testes. These extratesticular Leydig cells had characteristics of the active steroid-producing cell and seemed to be another source of testosterone.

Aspectos morfológicos do funículo espermático do préa (Galea spixii spixii) / Morphological aspects of the funiculus spermaticus of the préa (Galea spixii spixii)

Estudando os aspectos morfológicos de 14 pares de funículos espermáticos de preá (Galea spixii spixii), adultos, observa-se histologicamente, em quatro pares, que os seus componentes acham-se envolvidos por delgada cápsula de tecido conjuntivo frouxo recoberta pelo mesotélio. Sob esta cápsula, envolvendo completamente o funículo, encontra-se densa camada de tecido adiposo. Seus componentes estäo envolvidos por tecido conjuntivo frouxo. A artéria testicular mostra-se sempre contornada pelas veias testiculares, e possui: moderada túnica média, cujas fibras musculares lisas säo mantidas por uma orgenada rede de fibras reticulares; túnica interna constituída por endotélio, tecido conjuntivo subendotelial e nítida lâmina elástica interna; e a túnica externa, representada essencialmente por fibras colágenas com raras fibras reticulares e elásticas, que se confundde com a adventícia das veias e com o tecido conjuntivo intervascular. As veias testiculares formam o plexo pampiniforme, apresentam lumens amplos e irregulares, calibres variáveis, com paredes constituídas praticamente de endotélio vascular, desprovidas de válvulas e apresentam íntima relaçäo com a artéria. O segmento da artéria testicular obtido com Neoprene látex "450", em 10 moldes, exibe trajeto sinuoso, e como comprimentos médio, máximo e mínimo, respectivamente, 14,57cm, 16,60cm e 12,20cm à direita e 13,22cm, 12,20cm, e 11,70cm à esquerda; valores que näo apresentam diferenças significantes, ao nível de 5 por cento.

Morphological aspects of the funiculus spermaticus of the golden hamster (Mesocricetus auratus)

In a morphologic study of 43 pairs of spermatic cordsof adult Golden hamsters (Mesocricetus auratus), histology showed that in three pairs their components were covered by a thin mesothelial capsule. Under this capsule there was a dense layer of adipose tissue completely surrounding the spermatic cord. The componentswere covered by loose connective tissue showing a prevalence of a collagen fibers among reticular and elastic fibers. The testicular artery varied in diameter and showed a thick tunica media with muscle fibers sustained by a network of reticular fibers; the tunica interna consisted of endothelium, subendothelial connective tissue and a well-defined internal elastic membrane; the tunica externa or adventitia was formed by connective tissue, becoming a part of the adventitial layer of the testicular veins and intervascular connective tissue. The testicular veins formed the pampiniform plexus, which partially envelops the artery, have a wide and irregular lumina, thin walls made up almost exclusively of endothelium, have no valves and show a conspicuous relationship with the artery. The deferent duct was peripheral to the subcapsular adipose tissue, which partly surrounded it, and was joined by arterioles, venules, lymphatics and nerves. The segment of testicular artery obtained with the use of Neoprene latex "450" from 80 samples, corresponding to 40 pairs of spermatic cords, showed a winding course and a medium, maximum, and minimal lenght of 3.97, 6.2 and 2.4 cm, respectively, with a standard left and righ deviation of 3.88,5.7 and 1.8. These values showed no significant differences at the 5(per cent) level.

Análisis histomorfométrico del testículo neonatal / Histomorphometric analysis of neonatal testicle

La finalidad de este estudio fue cuantificar, en biopsias de tejido testicular neonatal, los histomorfométricos de mayor trascendencia. El estudio fue prospectivo, observacional y transversal. Se ingresaron neonatos masculinos recién fallecidos, exentos de patología gonadal o genética, a quienes inmediatamente después de la muerte se tomó biopsia testicular por punción escrotal mediante aguja Trucut Núm. 14. El material obtenido se fijó en solución de Bouin, se incluyó en parafina, se cortó a cuatro micras y se tiñió con hematoxilina y eosina. El análisis se efectuó con un videomicroscopio Hitachi con monitor de alta resolución y microfotografía. Se cuantificaron parámetros ya previamente descritos y algunos novedosos propuestos por los autores; de cada uno de ellos se obtuvo un promedio por testículo y, finalmente, un promedio con desviación estándar (DE) general. De las 13 muestras obtenidas sólo nueve fueron de utilidad. Los parámetros obtenidos fueron: 1) Recuentos de cordones seminíferos: a) cordones por campo de 100 000 micras cuadradas: 13.19, DE 4; b) diámetro cordonal: 58 micras, DE 6; c) superficie cordonal: 4 626 micras cuadradas, DE 473; d) densidad cordonal por campo de 100 000 micras cuadradas: 0.59, DE 0.13. 2) Recuentos de estirpes celulares: e) células germinales por cordón: 2.95, DE 0.8; f) índice de Mancini: 148 DE 39; g) índice de fertilidad tubular: 97 por ciento, DE 0.08; h) diámetro de célula germinal: 12 micras, DE 0.7; i) índice de células de Sertoli: 34, DE 13; j) longitud de células de Sertoli: 7 micras, DE 0.6. 3) Medición de tejido intersticial: k) espacio intercordonal: 2.8 micras, DE 1. Se concluye que el testículo neonatal muestra características bien definidas que hacen posible su estudio histomorfométrico mediante los parámetros descritos

Morphological studies on prespermatogonia and pre-Sertoli cells in the testes of 6- to 11-day-old golden hamsters.

The developmental progression of prespermatogenesis in the testes of 6- to 11-day-old golden hamsters has been studied by means of light and electron microscopy. The solid testicular cords prove to be built up by germ and supporting (pre-Sertoli) cells. The germ cells are present as centrally placed T1-prespermatogonia on days 6-7. Subsequently they develop into T2-prespermatogonia, shifting towards a more peripheral position within the testicular cords. From day 10 onwards, most of the germ cells are A-spermatogonia now occupying a marginal position within the cords. The supporting cells still proliferate during this period and can be subdivided into two distinct cell types, light-staining and dark-staining supporting cells. The numerical proportion of the two cell types continuously changes in favour of the light-staining ones. These light supporting cells appear to maintain a conspicuous spatial relationship to the germ cells throughout the whole developmental period observed here. The significance of the two types of supporting cells is discussed. The topographical arrangement of germ cells and light supporting cells, as found in the 6- to 11-day-old golden hamster testes, could imply a possible functional role in the regulation of germ cell development.

Histogenesis of human extraparenchymal Leydig cells.

From 64 consecutive autopsies of patients with neither testicular nor hormonal pathology, 26 showed extraparenchymal Leydig cells, located mainly in the epididymis and in the spermatic cord. The ultrastructural study of these specimens plus those obtained from 2 patients affected with functional testicular tumors leads to the following conclusions: (1) The origin of ectopic Leydig cells is not interstitial Leydig cells having infiltrated the testicular nerves and migrated along them towards ectopic locations. (2) The ectopic Leydig cells are considered to develop from undifferentiated precursor cells, located extraparenchymally, mainly inside and beside the testicular nerves. These precursor cells are similar to those observed in the testicular interstitium and have an ovoid shape and some cytoplasmic projections. The cytoplasm contains vesicles of smooth endoplasmic reticulum, lysosomes, lipid droplets and abundant microfilament bundles. The transformation from these cells into mature Leydig cells implies a progressive differentiation of the cytoplasmic components involved in steroid biosynthesis.

[Ultrastructure of human heterotopic Leydig cells]. / Die Ultrastruktur heterotoper Leydigzellen beim Menschen.

It was demonstrated by light and electron microscopical methods that Leydig cells also exist outside of the testis. In the spermatic cord and near the intraabdominal testicular vessels cells were observed with the characteristics of intratubular Leydig cells. Such characteristics were intranuclear precrystalline inclusions, mitochondria with tubular interior structures and lipid droplets, and a well developed smooth and rough endoplasmatic reticulum. The last two items can be regarded as signs of hormonal activity. The extratesticular Leydig cells in the spermatic cord are located within nerves that contain myelinated axons forming monoaxonal Schwann cell units. Possible they are sensory nerves originating in the receptors of the tunica albuginea. Synapses between axons and heterotopic Leydig cells were not found. The degree of the cytological differentiation leads to the conclusion that these cells may serve, for instance after orchidectomy, as substitutes for the Leydig cells inside the testis.