Active immunization of boars against gonadotrop in releasing hormone. I. Effects on reproductive parameters

Sexually mature boars were actively immunized against gonadotropin releasing hormone (GnRH) to characterize endocrine and gametogenic changes associated with immunoneutralization of endogenous GnRH. Injections of GnRH conjugated to bovine serum albumin (BSA) given five times over 24 wk induced production of antibodies against GnRH in all animals (n = 5). Active immunization against GnRH reduced serum concentrations of testosterone (P < 0.05) and luteinizing hormone (LH) (P < 0.05), testes volume (P < 0.01), paired testis weights (P < 0.05), paired epididymis weights (P < 0.05), sperm per testis (P < 0.01) and seminiferous tubule diameters (P < 0.001) when compared with controls (n = 4). These results indicate that both steroidogenic and spermatogenic functions are impaired in testes of mature boars actively immunized against GnRH.     

Seasonal changes in spermatogenic cell degeneration in the seminiferous epithelium of adult Japanese macaques (Macaca fuscata fuscata)

The degenerating pattern of spermatogenic cells in the seminiferous tubule of Japanese macaques was studied to clarify a relationship between seasonal changes of reproductive performances and cytological findings in the Japanese macaque. For light microscopy, testis samples were obtained from five adult animals by biopsy in April (nonmating season) and October (mating season). For electron microscopy, specimens from four additional macaques were used. Degenerating cells were found in all steps of spermatogenesis. In stages I to V of the cycle of the seminiferous epithelium, morphologically atypical pachytene spermatocytes were observed in 14.7 and 10.0% of the cells in the nonmating and mating seasons, respectively, although the difference in percentage was not significant. Mature spermatids with atypical features in those stages occupied 59.6% of the cells in the nonmating season, which significantly decreased to 34.1% in the mating season. These results imply that the seasonal change of sperm production is related, at least in part, to the process of degeneration of the spermatogenic cells in this species.     

Development of the seminiferous tubules after neonatal hemicastration in the boar

Development of the prepubertal seminiferous tubules of the right testis was characterized morphometrically every 14 days from 10 to 122 days of age in intact boars (I) and boars hemicastrated (HC) on Day 10 of life from two herds (Trial 1 and Trial 2). Comparisons were made between the remaining testis of Group HC boars and one testis in Group I boars. By 38 days of age seminiferous tubule length in Group HC boars was double (P less than 0.0001) that in Group I boars. Seminiferous tubule length did not differ between trials within treatments. The diameter of the seminiferous tubule was similar in Group HC and I boars but was greater (P less than 0.05) in Trial-1 than Trial-2 boars from Day 80 to 122 of life. Relative mass (mass of tissue/body mass) of Sertoli cells became 2-fold greater (P less than 0.0001), in Group HC than in one testis of Group I boars by 38 days of age and this difference was maintained throughout the experimental period. The relative mass of Sertoli cells was greater (P less than 0.05) in Trial-1 than Trial-2 boars within each treatment between 80 and 122 days of age. The relative mass of gonocytes was similar for all groups and treatments of boars. By 122 days of age the relative mass of spermatogenic cells was greater (P less than 0.05) in Group HC than in one testis of Group I boars and greater (P less than 0.01) in Trial-1 than Trial-2 boars within each treatment. Onset of spermatogenesis was first observed at 80 and 94 days of age in boars in Groups HC and I, respectively. Development of seminiferous tubule lumen was first observed at 94 and 108 days of age in boars in Groups HC and I respectively. Seminiferous tubule lumen, taken as a measure of fluid secretion of the Sertoli cells, occupied a greater (P less than 0.01) portion of seminiferous tubule in Trial-1 than Trial-2 boars within each treatment at the end of the experimental period. It is concluded that neonatal hemicastration of boars rapidly caused a compensatory seminiferous tubule elongation apparently due to Sertoli cell proliferation and an earlier onset of spermatogenesis. However, the gonocytes do not proliferate until they transform into spermatogonia.     

Impact of season on seminal characteristics and endocrine status of adult free-ranging African buffalo (Syncerus caffer)

Pituitary, gonadal and adrenal activity were compared in free-living, adult African buffalo bulls during the breeding and nonbreeding seasons. Frequent blood samples were collected for 2 h from anaesthetized bulls treated intravenously with saline, gonadotrophin-releasing hormone (GnRH, 200 micrograms), human chorionic gonadotrophin (hCG, 10,000 i.u.) or adrenocorticotrophic hormone (ACTH, 1.5 mg). Electroejaculates also were collected from anaesthetized bulls during the breeding and nonbreeding seasons. Pretreatment testosterone concentrations among bulls varied more during the breeding (0.17-23.0 ng/ml) than the nonbreeding (0.15-2.21 ng/ml) season. The variation within the breeding season was attributed to 8 of 25 bulls producing higher (P less than 0.05) serum testosterone (High-T; 16.28 +/- 2.03 ng/ml) and testicular LH receptor (1.53 +/- 0.22 fmol/mg testis) concentrations compared with their seasonal counterparts (Low-T; 0.95 +/- 0.26 ng/ml; 0.38 +/- 0.04 fmol/mg) or with all bulls during the nonbreeding season (0.90 +/- 0.27 ng/ml; 0.31 +/- 0.04 fmol/mg). The magnitude of GnRH- and hCG-induced increases in serum testosterone was similar (P greater than 0.05) between Low-T bulls and bulls during the nonbreeding season. In the High-T animals treated with GnRH or hCG, serum testosterone did not increase, suggesting that secretion was already maximal. Peak serum LH concentrations after GnRH were greater (P less than 0.05) in bulls during the nonbreeding than the breeding season; FSH responses were similar (P greater than 0.05). ACTH treatment did not increase serum cortisol concentrations above the 2-fold increase measured in bulls treated with saline, hCG and GnRH (P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structure of seminiferous tubules in antarctic seals

Summary The fine structure of seminiferous tubules from 5 crabeater, 2 leopard and 2 Ross seals showed that during the nonbreeding season the tubules were essentially similar in possessing spermatogenic and Sertoli cells. However, the tubules of leopard and Ross seals had more primary and secondary spermatocytes and spermatids than the crabeater seals. In general, the tubules were devoid of spermatozoa. The spermatids showed stages of maturation such as Golgi phase of acrosome formation, acrosomal cap formation and condensation of nuclei. Some spermatids degenerated in tubules. Both maturing and degenerating spermatids were closely associated with Sertoli cells. Junctional complexes with plaques of filaments were observed between Sertoli cells and the spermatogenic cells. Sertoli cells, irregular and polygonal, contained highly convoluted nuclei, strands of rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi complexes, small mitochondria, variable amounts of lipid droplets, lysosomes, lipofuscin granules and highly plicated plasma membranes. In brief, the spermatogenic activity had practically ceased in the testes and the animals probably secreted low levels of testosterone during the nonbreeding season.This research was supported in part by National Science Foundation Grants G.U. 30270 and G.U. 29829X from the Office of Polar Program and by NIH Grant 5 R01 AM11-376     

Distribution of type A spermatogonia in the mouse is not random

The distribution of type A spermatogonia was studied using drawings of cross-sectioned tubules at various stages of the spermatogenic cycle of perfusion-fixed, epoxy-embedded mouse testis. Spermatogonia were classified as either positioned opposite the interstitium or opposite the region where two tubules make contact or in a defined, intermediate region at which the two tubules diverged. At stage V, the population of type A spermatogonia, comprised of A(s) through A(al) cells, is randomly positioned around the periphery of the seminiferous tubule. The A(s) through A(al) population becomes nonrandomly distributed beginning at stage VI, being located primarily in regions where the tubule opposes the interstitium, and remains nonrandom through stage III of the next cycle. The A(1) spermatogonia of stage VII, derived from most A(pr) and A(al) spermatogonia, and the A(2) spermatogonia of stage IX, derived from the A(1) spermatogonia, are also nonrandomly positioned opposing the interstitium. However, the A(3) population of stage XI becomes randomly distributed around the tubule. To our knowledge, these are the first data to show that the more primitive spermatogonial types (A(s) to A(al)) move to specific sites within the seminiferous tubule. Division of the regularly spaced, more primitive spermatogonia (A(s) to A(al)) leads to the spread of their progeny (A(1) to A(4)) laterally along the base of the seminiferous tubule. The lateral spread from more or less evenly spaced foci ensures that spermatogenesis is conducted uniformly around the entire tubule. The data also suggest that the position of a seminiferous tubule in the mouse is stabilized in relationship to other seminiferous tubules.     

Effect of vascular endothelial growth factor and testis tissue culture on spermatogenesis in bovine ectopic testis tissue xenografts

Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation. To test this hypothesis, bovine testis tissue was treated with vascular endothelial growth factor (VEGF) at the time of grafting or explant cultured for 1 wk prior to grafting. For the VEGF experiment, 8-wk donor tissue and graft sites were treated with 1 microg of VEGF in order to increase angiogenesis at the graft site. For the testis tissue culture experiment, 4-wk-old donor testis was cultured for 1 wk prior to grafting to stimulate spermatogonial stem cell proliferation. Testis tissue grafts were removed from the mice 24 wk after grafting. VEGF treatment increased graft weight and the percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue.     

粗糙沼虾精巢发育的组织学

利用光镜技术,对粗糙沼虾精巢发育进行了研究,根据精子发生过程中每种生殖细胞所占的比例和发生的次序,并结合精巢的形态特征,把精巢发育过程分为五个时期,即精原细胞期,精母细胞期,精细胞期,成熟精子期及退化期,精原细胞期,精巢小,透明乳白色,生精小管内的生殖细胞以精原细胞为主;精母细胞期;精巢体积增大,半透明乳白色,主要由处于初级精母细胞的次级精母细胞阶段的生殖细胞组成;精细胞期,精巢体积继续增大,颜色加深,生精小管内的生殖细胞以精细胞为主;成熟精子期,精巢体积可达最大,紫红色,生精小管内充满着成熟的精子,退化期;精巢体积减小,半透明乳白色,生精小管内的成熟精子几乎排空。     

Quantitative studies of compensatory testicular hypertrophy following unilateral castration in the boar

Unilateral castration of Large White X Landrace boars at monthly intervals up to 5 months of age, with the remaining testis being removed 2 months later, resulted in compensatory hypertrophy of the testis which decreased with age. In pigs 3 and 4 months old there was significant hypertrophy of the testis but at 5 and 7 months of age testicular weight of the hemicastrates did not differ significantly from control values. The increase in the testicular weight of unilaterally castrated pigs was correlated with an increase in the number of Sertoli and germ cells at 3 months of age and germ cells at 4 months of age occupying the seminiferous epithelium. This was correlated with increased total seminiferous tubule length and larger cross-sectional area of the tubule. Sertoli cell occupancy did not differ significantly between unilaterally castrated and intact boars.     

Consequences of epididymal ligation on prepubertal rats

OBJECTIVE: To study prospectively experimental effects of unilateral epididymal obstruction on testis and epididymis histopathologically in prepubertal rats. STUDY DESIGN: Organ weights, mean seminiferous tubule diameter (MSTD), mean ductus epididymis diameter (MDED), mean tubular biopsy scores (MTBS) and histopathology of 21 male albino Wistar rats were compared with the immunostaining affinity of anti-desmin, anti-vimentin, anti-laminin and anti-collagen antibodies. RESULTS: Statistical analysis of mean weight of testes, MTBS, mean epididymal weight, MDED and MSTD was significant. Evaluation of testis and epididymis revealed cellular damage, basal membrane impairment and granulomatous infiltration. CONCLUSION: Histopathologic alterations with MSTD are early indicators for ipsilateral and contralateral injury. Even the presence of criteria such as intactness and cellular integrity does not guarantee there is no sperm leakage or immunologic damage. Therefore the impairment of basal integrity is a switch code in determining spermatogenic failure.     

Neonatal treatment with naloxone increases the population of Sertoli cells and sperm production in adult rats

Endogenous opioid peptides play an important role in the ontogenesis of the functional and morphological parameters of the seminiferous epithelium. The aim of this study was to evaluate the effects of neonatal manipulations with naloxone, an opioid antagonist, on the population of Sertoli cells and on sperm production in adult rats. Rats were assigned to receive 8 mug per gram of body weight twice a day with interval of 8 h of naloxone and they were compared to a control group receiving saline. Naloxone groups presented the following findings when compared to the control group: increased body weight from the 2nd to the 27th day; a smaller seminiferous epithelium height, smaller seminiferous tubule diameter, increased number of Sertoli cells and daily sperm production per testis, increased daily sperm production per gram per testis and increased total length of the seminiferous tubule of the treated groups. According to our study, the neonatal treatment with naloxone during the critical period of testis development was able to change the proliferative dynamics of Sertoli cells by an intra and/or extra testicular blockage of opioid receptors, confirming the direct relation between the number of Sertoli cells and the number of spermatozoids.     

Seasonal variation in pituitary-gonadal function in free-ranging impala (Aepyceros melampus).

Blood, testicular biopsies and electroejaculates were collected from adult male impala, free-ranging in the Kruger National Park (Republic of South Africa), during the breeding (rut; April-May) and nonbreeding (September-October) seasons. Blood samples were collected at 5-min intervals for 120 min from anaesthetized males (n = 7 impala/group) treated intravenously with saline, gonadotrophin-releasing hormone (GnRH: 1 microgram/kg body weight) or human chorionic gonadotrophin (hCG: 10 or 30 iu/kg). Semen was collected from six more animals during the breeding season and 12 animals during the nonbreeding season using a standardized electroejaculation protocol. Ejaculates obtained during the nonbreeding season were of inferior quality to those collected during the breeding season, and were characterized by lower sperm concentrations, poorer sperm motility and more morphologically abnormal sperm forms. Within season, there were no differences in testosterone secretion between the two hCG doses, and these responses were similar to those observed after GnRH, but during the rut, testosterone secretion stimulated by both GnRH and hCG was approximately nine times greater than during the nonbreeding season. This seasonal increase in testosterone production was associated with a doubling in testicular volume and concentrations of luteinizing hormone (LH) receptors. Although concentrations of testicular follicle-stimulating hormone (FSH) receptors were similar between seasons, receptor content increased during rut as a result of increased testicular volume. In contrast to testosterone secretion, basal LH and FSH secretions were unaffected by season and GnRH-induced gonadotrophin secretion was reduced during rut.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes in the testicular and antler cycles of reindeer, Rangifer tarandus

A total of 111 male reindeer of various ages was shot in all months of the year to study the relationship between the seasonal changes in testicular activity and the antler cycle. From the changes in testis weight, seminiferous tubular tissue area and plasma testosterone values and the occurrence of spermatogenesis, it is concluded that calves attain physiological puberty in their first year, during which they also complete an antler cycle. The amplitude of the cyclical change in testis weight and plasma testosterone values increases with age and can be correlated with the earlier onset of events in the spermatogenic and antler cycles of older animals. The duration of the spermatogenic and testosterone cycles of reindeer is short, and is inversely related to the long period spent without antlers. It is suggested that testosterone strongly influences the antler cycle of reindeer males.     

Apoptosis during spermatogenesis: the thrill of being alive

Emerging clues about the apoptotic molecular mechanisms operating during spermatogenesis indicate that the activation mechanism of executioner caspases may diverge from the traditional signaling operating in the immune system. Two issues are now been debated: (1) Whether the massive apoptosis of spermatogenic cells observed during the first spermatogenic wave represents a mechanism for ensuring the steady state sperm output in a given segment of a seminiferous tubule. (2) Whether apoptosis just represents a local remodeling process commanded by the Sertoli cell constraints to satisfy the differentiation needs of a juxtaposed cellular domain of spermatogonial, spermatocyte, and spermatid cohorts in the adult testis.     

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.     

Effects of a single ethanol injection into the vas deferens on the testicular function of rats.

1. A new approach to rapid male sterilization has been studied by giving a single injection of 95% ethanol directly into the vas deferens. It produced an effective block in the lumen. The mating exposure test showed that the males were sterile. The sperm granulomas at the site of vas injection were confirmed. 2. Ethanol injection in the vas deferens caused an atrophy of the testes. Extensive necrosis and exfoliation of the seminiferous elements were conspicuous. 3. Decrease in testicular weight, seminiferous tubule diameter and RNA and sialic acid levels after 4 weeks of vas injection were associated with the histological evidence for severe degeneration of spermatogenic elements. The protein contents of testis, epididymides and seminal vesicles did not change. 4. Testicular cholesterol, total lipids and alkaline phosphatase activity was increased. 5. Low sialic acid levels in the testis, epididymides and seminal vesicles of vas-injected rats indicated an inhibition of androgen production, which was further reflected in reduced nuclear diameters of leydig cells.     

Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules

The mule (Equus mulus mulus) is a sterile hybrid domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). Usually, spermatogenesis in mules does not advance beyond spermatocytes. In the present study, we performed a comparative and more accurate morphometric and functional investigation of the testis in donkeys and mules. Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells, the total length of seminiferous tubules in mules was significantly smaller than in donkeys. However, the percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules was approximately 95%. The total number of Sertoli cells per testis observed in donkeys and mules was very similar. However, the total number of Leydig cells in mules was approximately 70% lower than in donkeys. At least in part, this difference was probably related to the lower number of germ cells present in mule seminiferous tubules. Although spermatogenesis in mules did not advance beyond secondary spermatocytes/newly formed round spermatids, germ cell associations in the seminiferous epithelium and pachytene spermatocytes nuclear volume in donkeys and mules were similar. The duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Each spermatogenic cycle in donkeys lasted 10.5 days. A similar value was found in mules ( approximately 10.1 days). Considering that the entire spermatogenic process takes approximately 4.5 cycles to be completed, its total duration in donkeys was estimated to last 47.2 days. The results found for mules suggest that the mechanisms involved in the determination of testis structure and function are probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis. In this regard, this species is a potential model for transplants of germ cells originated from donkeys and horses or other large animals.     

Immunohistochemical Localization of Mitochondrial Fatty Acid ��-Oxidation Enzymes in Rat Testis

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid β-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid β-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid β-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis. (J Histochem Cytochem 58:195–206, 2010)     

Spontaneous degeneration of germ cells in normal rat testis: assessment of cell types and frequency during the spermatogenic cycle.

The occurrence of degenerating germ cells in the cycle of the seminiferous epithelium was measured in testicular tissues from eight normal adult rats. Testes were perfusion fixed, embedded in epoxy resin and, after sectioning a total of 180 randomly selected blocks at 1 microns, stained sections were examined by light microscopy; all cross-sectioned seminiferous tubules were categorized into one of 14 stages of the spermatogenic cycle. The number of degenerating cells per tubule was recorded in 2103 tubules. Degenerating germ cells were not detected at stages II-VI, and only rarely at stage VII (n = 366 tubules) in which one primary spermatocyte and one step 19 spermatid degenerated. All other stages exhibited a greater incidence of degenerative germ cells, particularly at stage XIV where, on average, the frequency of degenerating cells per round seminiferous tubule was about 40 times greater than at stage VII. The results indicated that, in the normal adult rat testis, the germ cells are least at risk of degeneration as they pass through stage VII.     

Postnatal sexual development of testis and epididymis in the rabbit: growth and maturity patterns of macroscopic and microscopic markers

We examined the macroscopic variables related to the size of testis and epididymis, and the microscopic variables related to the tissue composition of testis to determine the onset of the male reproductive activity.The present work was carried out using two genetic lines of rabbits showing different reproductive aptitudes to assess the effects of genetic line and birth season on age-related changes of the testes and epididymis.The Caldes and Prat genetic lines showed similar developmental profiles for most of the variables studied. The main changes in the development pattern were observed at younger ages. The Caldes genetic line presented a greater live weight and a smaller testicular volume that the Prat genetic line at any age. No differences in the studied microscopic variables were found between the two genetic lines, except in the variable percentage of seminiferous tubules with presence of lumen.A significant effect of the birth season was found in live weight, testis volume, epididymis volume, percentage of seminiferous tubules with presence of elongated spermatids and diameter of seminiferous tubules. The absolute values and the values relatives to its own value at the adult stage of the variables live weight, testis volume, epididymis volume and in variables related to the functional maturity were lower in animals born in the summer season. Volume growth for both testis and epididymis was delayed in animals born in the summer season.