Characterization of bovine transcripts preferentially expressed in testis and with a putative role in spermatogenesis

Although the number of genes known to be associated with bovine spermatogenesis has increased in the past few years, regulation of this biological process remains poorly understood. Therefore, discovery of new male fertility genetic markers is of great value for assisted selection in commercially important cattle breeds, e.g., Nelore, that have delayed reproductive maturation and low fertility rates. The objective of the present study was to identify sequences associated with spermatogenesis that could be used as fertility markers. With RT-PCR, the following five transcripts preferentially expressed in adult testis were detected: TET(656) detected only in adult testis; TET(868) and TET(515) expressed preferentially in adult testis but also detected in fetal gonads of both sexes; and TET(456) and TET(262). expressed primarily in the testis, but also present in very low amounts in somatic tissues. Based on their homologies and expression profiles, we inferred that they had putative roles in spermatogenesis. Detection of sequences differentially expressed in testis, ovary, or both, was a useful approach for identifying new genes related to bovine spermatogenesis. The data reported here contributed to discovery of gene pathways involved in bovine spermatogenesis, with potential for prediction of fertility. (C) 2011 Elsevier Inc. All rights reserved.

Pre- and postnatal exposure to ambient levels of urban particulate matter (PM(2.5)) affects mice spermatogenesis

This work characterizes the effects of ambient levels of urban particulate matter (PM(2.5)) from the city of Sao Paulo on spermatogenesis using mice exposed during the embryo-fetal and/or postnatal phases of development. Parental generations (BALB/c mice) were exposed to air pollution in chambers with or without filtering PM(2.5) for 4 months. Animals were mated, and half of the 1-day-old offspring were moved between chambers, which yielded prenatal and postnatal groups. Remaining offspring comprised the non-exposed and pre+postnatal exposed groups. After 90 days, the animals were sacrificed for testis collection and weighing. Optical microscopy was used for the morphometric analyses of the cell counts, spermatogenic cycle, proliferation, and apoptosis. Prenatally exposed animals presented reduced body and testicular weight with an increased gonadosomatic index (GSI). Testicular volume also decreased, as well as the tubular diameter in testes of the same animals. Proliferation, apoptosis, and spermatogenic cycle analyses showed no significant differences among groups. However, the tubules at stage VII of pre- and postnatal animals presented a reduced number of elongated spermatids. Pre+postnatal group presented higher spermatid head retention at stages VIII-XII. These results show that ambient levels of PM(2.5) from Sao Paulo city affect spermatogenesis by damaging sperm production.

Juvenile hormone III and nutrition effects on spermatogenesis in the 4th instar nymphs of Panstrongylus megistus (Hemiptera: Reduviidae)

When 4th instar nymphs of Panstrongylus megistus are fed with a saturant blood meal, there is an intense proliferation of the spermatogonia. At the end of the intermoult, the older spermatogonial cysts differentiate into 1st primary spermatocyte cysts. In the nymphs deprived of the blood meal this evolution is not observed, but a small growth of the testicular follicles occurs, due to a few mitotic divisions. This growth is observed at least, until 25 days after ecdysis. Since day 15, an autolytic process starts in the older spermatogonial cysts. The presence of exogenous juvenile hormone III (JH III) does not promote the development of the germ cells in the fasting insects. There is only a small growth of the testicular follicles and the autolytic process is also observed. In the precocious adults obtained by allatectomy or precocene II treatment, germ cells are observed in all development stages, except packed and elongated spermatozoa bundels.

Occurrence of "Nuages" and "Lamellae Anulata" during spermatogenesis in Dermatobia hominis (Diptera: Cuterebridae)

Various types of "nuages" and "lamellae anulata" can be found during Dermatobia hominis spermatogenesis. In spermatogonia, the "nuages" occur as granules juxtaposed to the cytoplasmic face of the nuclear envelope or as cytoplasmic granules similar to glycogen granules. In spermatocytes, in addition to the "nuages", dense spherical bodies of approximately 1.0 µm in diameter are also observed. In the spermatids the "nuages" can be of the following types: perinuclear granules, spherical granules with diameters varying in length from 0.5 to 1.0 µm, granules similar to glycogen granules, granules with variable diameters which accumulate at the flagellum base forming the centriole adjunct, or remain in the cytoplasm. "Nuages" can also be observed in these cellular types as dense masses, without a definite outline and are common to animal germinal cells in general. The "lamellae anulata" on the other hand, are observed only in spermatocytes I and in early spermatids, being always immersed in electron-dense material of indefinite outline. In spermatids, the "lamellae anulata" are close to the nuclear envelope suggesting, in spite of opposing opinions, that these cells are envolved in the synthesis and transport of material from the nucleus to the cytoplasm.

Spermatogenesis of the Spider Crab Maja brachydactyla (Decapoda: Brachyura)

This study describes spermatogenesis in a majid crab (Maja brachydactyla) using electron microscopy and reports the origin of the different organelles present in the spermatozoa. Spermatogenesis in M. brachydactyla follows the general pattern observed in other brachyuran species but with several peculiarities. Annulate lamellae have been reported in brachyuran spermatogenesis during the diplotene stage of first spermatocytes, the early and mid-spermatids. Unlike previous observations, a Golgi complex has been found in midspermatids and is involved in the development of the acrosome. The Golgi complex produces two types of vesicles: light vesicles and electron-dense vesicles. The light vesicles merge into the cytoplasm, giving rise to the proacrosomal vesicle. The electron-dense vesicles are implicated in the formation of an electron-dense granule, which later merges with the proacrosomal vesicle. In the late spermatid, the endoplasmic reticulum and the Golgi complex degenerate and form the structures–organelles complex found in the spermatozoa. At the end of spermatogenesis, the materials in the proacrosomal vesicle aggregate in a two-step process, forming the characteristic concentric three-layered structure of the spermatozoon acrosome. The newly formed spermatozoa from testis show the typical brachyuran morphology.

Ultrastructure of spermatogenesis and sperm development in Saccocoelioides godoyi Kohn & Froes, 1986 (Digenea, Haploporidae)

Ultrastructural observations of spermatogenesis and sperm development of Saccocoelioides godoyi, an intestinal parasite of Leporinus friderici (Bloch, 1794) are described. The irregular-shaped spermatogonia form a peripheral layer, and show a prominent nucleus. Spermatocytes are larger than spermatogonia, and in the early stage present synaptonemal complex. Spermatids show nuclei smaller than the spermatocytes. Spermiogenesis is characterized by outgrowth of the zone of differentiation, presenting basal bodies, separated by an intercentriolar body. At the end of this process, the spermatozoa are released into the residual cytoplasmic mass. The spermatozoa of S. godoyi are elongate, similar to the pattern described for other Digenea, showing nuclei, mitochondria and two axonemes with the 9+1 configuration. The peripheral cortical microtubules on the dorsal and ventral faces are laterally interrupted.

Spermatogenesis and spermiogenesis in Didymocystis wedli Ariola, 1902 (Didymozoidae, Digenea)

The ultrastructure of the male reproductive system of Didymocystis wedli was studied for the first time, demonstrating spermiogenesis and spermatogenesis at different cell stages. The spermatozoa morphology was compared with that of other Digenea species. It was observed that the different cells of the spermatogenesis process follow the classic pattern reported for the majority of the parasitic platyhelminthes. During spermiogenesis, rootlet fibers, electrondense bodies and median cytoplasmic process were not observed. The mature spermatozoa of D. wedli were filiform, presenting nucleus, mitochondrion and two 9+1 axonemes, with a biflagellate distal extremity.

Relationship of mating systems and metabolic rate with cycle length of spermatogenesis and sperm production rate in shrews

SUMMARY : The shrews are among the most ancient of living eutherian mammals. They represent an interesting comparative model because of their extreme divergent species. The two shrew subfamilies, Soricinae and Crocidurinae are characterized by fundamental differences concerning their metabolic rates, litter size, period of gestation and different mating pattern. In this study we established and compared the sperm characteristics in four species of different genera of shrews (Sorex araneus, Neomys fodiens, Crocidura russula and Suncus murinus) in the context of the sperm competition hypothesis. The sperm competition concerns the competition between ejaculates of different males for fertilization of ova of a female within a single estrus period. As expected, a greater relative testis size (indicating the importance of polyandry) was associated with a higher number of cauda epididymal spermatozoa, higher level of circulating testosterone and a higher percentage of progressive sperm motility. In addition, we investigated if the basal metabolic rate (BMR) and relative testis size (RTS) may be correlated with the cycle length of spermatogenesis. In this purpose, we determined and compared the cycle length of spermatogenesis in six species of shrews belonging to two subfamilies: Soiricinae (Sorex araneus, Sorex coronatus, Sorex minutus, Neomys fodiens) and Crocidurinae (Crocidura russula, Sunctes murinus). Our results indicate that sperm competition and metabolic rate may act independently or together reducing cycle length of spermatogenesis and thus increase sperm production. We finally investigated this correlation across 32 mammalian species. After testing the data for phylogenetic independence, our results showed that BMR explained only 21 % of the variation, while the RTS explained 44% of the variation of the cycle length of spermatogenesis. The level of the sperm competition, indicated by RTS, is thus to our knowledge the most important factor influencing the speed of spermatogenesis in mammals. RESUME : Les musaraignes sont parmi les plus anciens mammifères vivants. Grâce à leurs extrêmes divergences, ils sont souvent utilisés comme modèles dans des études comparatives. Les deux sous-familles Soricinae et Crocidurinae sont caractérisées par des différences fondamentales, notamment en termes d'intensité du métabolisme, des stratégies de reproduction et du comportement social. Dans la première partie de cette étude, nous avons établi et comparé certaines "caractéristiques des spermatozoïdes chez quatre espèces de musaraignes appartenant à des genres différents (Sorex araneus, Neomys fodiens, Crocidura russula et Suncus murinus). Les résultats ont été interprétés dans le contexte de la théorie de la compétition spermatique, c'est-à-dire la compétition entre le sperme de deux ou plusieurs mâles pour féconder un maximum d'ovules de la même femelle. Cette compétition spermatique peut amener à certaines adaptations biologiques afin de produire plus de sperme. Comme attendu, une grande taille relative des testicules est associée à un nombre élevé de spermatozoïdes, dont la majorité présente une mobilité progressive. Un taux élévé de testostérone a également été observé. De plus, nous avons étudié l'influence du métabolisme basal ainsi que l'intensité de la compétition spermatique sur la durée du cycle de la spermatogenèse. Dans ce but, nous avons déterminé et comparé les durées de la spermatogenèse chez six espèces de musaraignes appartenant à deux sous-familles : Soricinae (Sorex araneus, Sorex coronatus, Sorex minutus, Neomys fodiens) et Crocidurinae (Crocidura russula, Suncus murinus). Les résultats obtenus indiquent que ces deux facteurs (l'intensité du métabolisme basal et de la compétition spermatique) agissent d'une manière dépendante ou indépendante dans le même sens. La conséquence de ces actions est une diminution de la durée de la spermatogenèse entraînant une augmentation de la production de spermatozoïdes. Nous avons finalement étudié ce phénomène dans l'ensemble des mammifères. Après avoir testé l'indépendance phylogénétique, nos résultats montrent que l'intensité de la compétition spermatique indiquée par le RTS est mieux corrélée avec la régulation de la durée de la spermatogenèse qu'avec l'intensité du métabolisme.

Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

Effect of Pcbs (Aroclor 1254) on spermatogenesis In Testis Of Mouse

Polychlorinated biphenyls (PCBs) are a group of halogenated aromatic hydrocarbons, synthetic chemicals which do not occur naturally in the environment. PCBs are considered potential endocrine disruptors. They are estrogen-like and anti-androgenic chemicals in the environment contain potentially hazardous effects on male reproductive axis resulting in infertility and other hormonal dependent reproductive functions. These toxic substance cause alteration of the endocrine systems, mimic natural hormones and inhibit the action of hormones. The aim of this study is to examine the effect of Polychlorinated biphenyls (PCBs) on testicular development of male reproductive system in mice. The male mice were randomly assigned to five groups with each group comprising twenty-one members. In those mice were administered 0 μg/kg (control group) and 0.5, 5, 50, 500 μg/kg Aroclor 1254 (treated group) by gavages three time per week. Treatment was carried out for 50 days after which the mouse was sacrificed and the body weight, testicular weight; epedidymis weight, sperm mortality, sperm count and sperm abnormality were taken. However, there was no significant difference in testicular/body weight and epididymis/body weight ratio in treated group compared with the control group. According to the analysis of sperm quality, Aroclor 1254 treated group demonstrated significant increased in sperm mortality in 500 μg/kg; decreased the sperm count in 0.5 μg/kg, 5 μg/kg, 50 μg/kg and 500 μg/kg; and significantly elevate the sperm abnormality in 50 μg/kg and 500 μg/kg compared to the control in a dose-dependent manner. The sex hormone levels in the testes were detected by radio-immunoassay (RIA) method. The levels of testosterone and 17β-estradiol did not reveal significant alteration (p< 0.05) in PCBs treated groups compared to the control in a dose-dependent manner. The testis were obtained and subjected to routine histopathology following exposure to PCBs in supplement diet. The diameter of the seminiferous tubule and the number of Sertoli cells in the treated group increased significantly (p< 0.05) in comparison to the control group. For the spermatogenic cell, the number of germ cell in high concentration decreased significantly (p< 0.05). However, spermatogonia cells in PCB treated group showed non-significant difference (p< 0.05) compared to the control. vii Western blot analysis was used to determine the level of protein between the control and treated group. The level of Proliferating cell nuclear antigen (PCNA) was determined and the results have shown no significant alteration between the treated groups and the control. the level of sex hormone receptor (ER α/β); Androgen receptor (AR) were identified in the testes to detect the proliferative effect induced by PCBs. Statistical analyses of AR, ER α and ER β did not reveal significant difference between the control and the treated groups. In the present study, we continue to investigate adverse effect of Aroclor 1254 and their mechanism on spermatogenesis. The result of Sperm quality and histopathology showed that Aroclor 1254 at low concentration induce inhibitory effect on testicular function of male mouse.

Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing.

Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP.

Cycle length of spermatogenesis in shrews (mammalia: soricidae) with high and low metabolic rates and different mating systems.

The aim of the present study was to establish and compare the durations of the seminiferous epithelium cycles of the common shrew Sorex araneus, which is characterized by a high metabolic rate and multiple paternity, and the greater white-toothed shrew Crocidura russula, which is characterized by a low metabolic rate and a monogamous mating system. Twelve S. araneus males and fifteen C. russula males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determinations, we applied the classical method of estimation and linear regression as a new method. With regard to variance, and even with a relatively small sample size, the new method seems to be more precise. In addition, the regression method allows the inference of information for every animal tested, enabling comparisons of different factors with cycle lengths. Our results show that not only increased testis size leads to increased sperm production, but it also reduces the duration of spermatogenesis. The calculated cycle lengths were 8.35 days for S. araneus and 12.12 days for C. russula. The data obtained in the present study provide the basis for future investigations into the effects of metabolic rate and mating systems on the speed of spermatogenesis.

Germ cell-specific targeting of DICER or DGCR8 reveals a novel role for endo-siRNAs in the progression of mammalian spermatogenesis and male fertility.

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.