Process Of Spermiation Research Articles

Genes associated with cell modelling provides new insights into spermiation mechanism in Cyprinus carpio

Spermiation, an act of sperm release, depends on several molecular factors. Despite hormonal administration, spermiation failure is a primary concern in certain fishes. In this study, the molecular mechanisms of spermiation have been analyzed in Cyprinus carpio by comparative transcriptomics. Unigenes for C. carpio control (CCC), which were injected with PBS (Phosphate-buffered saline), and C. carpio treated (CCT), which were injected with ovatide, were 107,616 and 133,435, respectively. A total of 93 genes were identified as involved in the spermiation process, including those related to gonadal steroidogenesis, cell growth, cell adhesion, and cytoplasmic matrix formation. The cd63, CENPS, rasa1a, and genes for gonad steroidogenesis, cell growth, cell adhesion, and cytoplasmic matrix formation were analyzed. Gene expression analysis revealed tubulobulbar complexes mediated disengagement of spermatozoa and JAK2 signaling regulated cyst breakage in teleost for the first time. Analysis was done from the changes at the molecular level to the final act of spermiation. Tissue histology analysis was conducted in accordance with the molecular study, which showed structural changes. Induced breeding in fish plays a key role in seed production in aquaculture sector. However, there are several constraints the sector is still facing due to lack of extensive knowledge regarding the mechanisms of spermiation and species-specific response to hormonal dosage. This study is relevant to understand the molecular mechanisms involved in spermiation and the stages which mark as critical point of sperm release after administrating the inducing agent. This study also lays the groundwork for further exploration of species-specific responses to hormonal treatments, aiding sustainable seed production in the fisheries sector.

Single-cell RNA sequencing technology in human spermatogenesis: Progresses and perspectives.

Spermatogenesis, a key part of the spermiation process, is regulated by a combination of key cells, such as primordial germ cells, spermatogonial stem cells, and somatic cells, such as Sertoli cells. Abnormal spermatogenesis can lead to azoospermia, testicular tumors, and other diseases related to male infertility. The application of single-cell RNA sequencing (scRNA-seq) technology in male reproduction is gradually increasing with its unique insight into deep mining and analysis. The data cover different periods of neonatal, prepubertal, pubertal, and adult stages. Different types of male infertility diseases including obstructive and non-obstructive azoospermia (NOA), Klinefelter Syndrome (KS), Sertoli Cell Only Syndrome (SCOS), and testicular tumors are also covered. We briefly review the principles and application of scRNA-seq and summarize the research results and application directions in spermatogenesis in different periods and pathological states. Moreover, we discuss the challenges of applying this technology in male reproduction and the prospects of combining it with other technologies.

Recombinant vs purified mammal gonadotropins as maturation hormonal treatments of European eel males

In the past three decades the European eel Anguilla anguilla experienced up to 99% decline in recruitment in some parts of its distribution range, thus breeding in captivity is nowadays considered key in order to save this species. With this in mind, obtaining high quality gametes is fundamental, as is the ongoing study of new hormonal treatments in order to improve current methods. Therefore, the aim of this research study was i) to assess the effect of two hormonal treatments (OVI, a recombinant α-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine) on the reproductive performance of European eel males, and, after choosing the best hormone, ii) to compare the effects of three doses in order to cut the costs of artificial maturation.Our results indicated that the type of hormone used (recombinant vs purified gonadotropins) significantly affected the progression of spermiation in European eel males, and that the recombinant hormone (OVI) produced better results in terms of sperm quantity and quality in most of the weeks of the treatment, remaining thus an effective treatment to induce spermiation in this species. On the other hand, in terms of the doses experiment, our results showed that from the lowest to the highest dose (0.25 to 1.5 IU/g fish) all the treatments were able to induce the whole spermiation process. However, a weekly dose of 1.5 IU/g fish of recombinant hormone (OVI) was necessary in order to provide a notable amount (volume and density) of high quality (motility and velocity) samples throughout the treatment.Finally, the economic analysis demonstrated that the recombinant hormone (OVI, 1.5 IU/g fish) had a greater profitability than the other treatments, making it possible to obtain high-quality sperm for a lower price. In this context, and considering the fact that in the first few weeks of any hormonal treatment there is no high-quality sperm production, long-term hormonal therapies are necessary in order to lessen the cost of high-quality European eel sperm.

Cytology of the Pituitary Gonadotrophs, Histological Characteristics of Interrenal and Chromaffin Cells in Relation to Testicular Activities in Mystus Vittatus (Siluriformes, Bagridae) During Growth, Maturation and Spawning Phases

Abstract The histological changes observed in the pituitary corticotrophs, gonadotrophs, adrenocortical tissues and testicular cells in M. vittatus (Bloch, 1794) have been studies during growth, maturation and spawning phases. The studies based on the changes observed in the cell types, shape and size of the cells of the adrenocortical tissues, testes and the overall percentage of gonadotroph (GTH) and thyrotroph (TSH) cells of the pituitary. However, during growth phase, in proximal pars distalis (PPD) the considerable increment of GTH and TSH have been observed having intense aniline blue stain. The corticotrophs (ACTH) also showed significant accumulation of fuchsinophilic cytoplasmic granules. The cytoplasmic features and the architecture of the interrenal cells were well coincident with the increase of different spermatogenic cells. During the maturation phase dense granulation in the GTH and TSH cells appeared to be concomitant with the spermiation. The amount of cytoplasmic granules of the interrenal cells increased than chromaffin cells and was well coincidence with the increase of spermatids and spermatozoa. The hyperactive and vacuolated features of the interrenal cells during spawning phase appeared to be concomitant with the final process of spermiation.

Effect of thermal regime on fatty acid dynamics in male European eels (Anguilla anguilla) during hormonally-induced spermatogenesis

Abstract Little is known about the role of fat and fatty acids in European eel spermatogenesis. The aim of this research was to study the changes in fat content and to carry out a quantitative analysis of the fatty acid composition of the muscle, liver and gonad of European male eels during hormonally induced sexual maturation. Three different thermal regimes were used (two variable: T10 and T15; and one constant: T20) to replicate the changes in temperature that these fish experience during their transoceanic reproductive migration. Spermatogenesis was reached earlier in treatment T20, suggesting that spermatogenesis in the European eel is closely regulated by water temperature. Although eels lose body mass due to the period of fasting that accompanies gonadal growth, no significant changes were found in the fat and fatty acid content of the muscle during the experimental period. With regard to the liver, the levels of palmitic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids increased significantly at the start of the spermiation process in all the thermal treatments. In the testis, levels of EPA, arachidonic acid (ARA) and DHA remained constant during the maturation process whereas the levels of the rest of the fatty acids decreased significantly. The stability of the ARA and EPA levels in the testis may have a physiological significance, whereas the stability of the DHA levels may have a structural significance. The results suggest that the progression of spermiation is influenced by water temperature and demonstrate the importance of EPA, ARA and DHA in European eel reproduction. This study demonstrates that complementary research focusing on the lipid composition of commercial diets could improve the sperm quality of this species.

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen.

There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90s), the thermal regimen (i.e. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30s is recommended as the standard time for motility acquisition. When eels were kept at 20°C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8±1.3%; mean ± s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0±1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.

Histomorphological Assessment of Spermatogenesis in African Catfish Clarias gariepinus with reference to Spermatogonial Proliferation

The fresh water catfish is the most predominant species of the teleost bred in captivity in Nigeria and has provided a model for the study of reproduction in aquatic vertebrates, but information on the number of mitotic divisions and spermatogonial generations remains inconclusive. This investigation aimed at assessing the number of generations of spermatogonia produced prior to meiotic division during spermatogenesis. Results indicate that spermatogenesis occurred in cysts and germ cells at the same stage of maturation formed clones round the Sertoli cell. Spermatogonia went through 5 rounds of mitotic divisions to produce 6 generations of B clones (B1-B6) before producing primary spermatocytes, secondary spermatocytes and haploid spermatids . The process of spermiation was not synchronized in all clones of the cysts. Cellular maturation and spermiation occurred in the most advanced clones. Each cyst appeared to simulate the spermatogenic process that occurs in the seminiferous epithelium of mammalian testis. It is concluded that spermatogenesis in captive bred African catfish Clarias gariepinus is cystic, and that spermatogonia arrangement in the cysts is unrestricted and progresses through 5 mitotic divisions to produce 64 spermatogonia type B , 100 mono-flagellated spermatozoa. Approximately, 68% germ cells complete spermatogenesis, following 32 % germ cell loss.

Differential Proteomic and Phospho-proteomic Analysis of Normal versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS

Spermiation is the final step of spermatogenesis and involves release of mature spermatids from the Sertoli cells. It is a complex process involving displacement and removal of spermatid cytoplasm, formation and degradation of tubulobulbar complexes and progressive loss of adhesive junctions, including ectoplasmic specializations and subsequent phagocytosis of residual bodies by the Sertoli cell. Spermiation occurs in stage VIII of the rat seminiferous epithelium cycle. In spite of the important role played by spermiation process in sperm output to the epididymis, very little is known about this process. To enhance our knowledge of the sperm release biology, we sought to understand the molecular events occurring at the time of spermiation. Towards this aim, we induced spermiation failure condition by 17β estradiol treatment to adult male rats and compared with the normal spermiation. On comparing both the groups, we identified a total of 104 differentially expressed proteins and 23 differentially expressed phosphoproteins by LC-MS/MS analysis and quantitation. Localization and expression of some of the validated differential proteins in the testes highlight their importance during the sperm release. The present study represents an initial step to understand the molecular basis of the process of spermiation.

Comparative Study of Sex Steroid Levels of Persian Sturgeon, Acipenser persicus Males in Responding Negative and Positive to LHRH-A2 Hormone

Gonadotropin-releasing hormone agonists (GnRHa or LHRHa) have been used extensively in order to stimulate the release of pituitary luteinizing hormone (LH) required to induce sexual maturation (e.g., spermiation). In this research, blood serum testosterone (T), 11-ketotestosterone (11-K) and progesterone (P4) levels were measured in Persian sturgeon, Asipenser persicus (Borodin, 1897), males during propagation season. The type and dose of hormone administration for artificial propagation were LH-RH-A2 and 5 μg kg-1, respectively. In males which responded by spermiating, serum steroids levels (T, 11-K and P4) were higher than in non spermiating males. Following hormonal stimulation, levels of all three steroids increased significantly in spermiting males 14h after LHRH- A2 injection. T levels increased slightly in non spermiting males and other steroids did not change during the spermiation process. The rise of T levels in both males show that this steroid as a dominant and key androgen related with maturation in Persian sturgeon.

Seasonal reproductive cycle of Waigieu seaperch (Psammoperca waigiensis)

Predictive and reliable parameters of reproductive status are integral aspects of sustainable fisheries and aquaculture management. These parameters are also important for an accurate evaluation of the effects of different treatments on sexual maturation in fish farming. In the present study, we have characterized the seasonal reproduction profile and described changes in sex steroids in relation to gonadal maturation and development in female and male Waigieu seaperch (Psammoperca waigiensis). The experimental period covered a full calendar year (January–December). In males and females, we observed that plasma sex steroid hormones [oestradiol-17β (E2), testosterone (T), 11-ketotestosterone (11-KT) and progesterone (P)] levels showed monthly fluctuations during the spawning period. Particularly, plasma steroid hormone levels were positively associated with gonadosomatic index values. In addition, high levels of plasma steroid coincided with recruitment of oocytes into yolk accumulation in females. The main spawning period occurred between April and October in females, and between March and November in males. The non-aromatizable androgen, 11-KT is generally believed to be the active male-specific androgen in teleosts, and is associated with the process of spermiation, development of secondary sexual characteristics and regulation of male reproductive behaviour in most teleost species. In this study, we found relatively high amounts of 11-KT in females between May and December, suggesting an integral role in the maturation process, also for the females. A rapid peak in plasma P level was observed in November and suggests significant roles during post-spawning and/or resting periods in both female and male fish. Furthermore, all oocyte developmental stages were present within the same sampling month and also within the spawning period, demonstrating the gamete group asynchronous developmental strategy. Overall, Waigieu seaperch showed strong seasonality in reproductive development with corresponding sex steroid patterns. The data presented in this study may contribute to the understanding of the reproductive endocrinology of a tropical marine finfish with increasing industrial prospects and sustainable aquaculture of this species in a developing country, such as Vietnam.

Structure and control of a cell-cell adhesion complex associated with spermiation in rat seminiferous epithelium.

Spermiation, the release of late spermatids from the Sertoli cell, is disrupted by a number of toxicants. Control of the spermiation process, and the proteins that interact to adhere mature spermatids to Sertoli cells, is poorly understood. In these studies we used immunohistochemistry, coimmunoprecipitation/Western blotting, and mass spectrometry to refine an earlier model of sperm adhesion proposed by our laboratory. We have identified specific proteins linked together as part of a multiprotein complex, as well as several additional proteins (cortactin, ERK1/2, and 14-3-3 zeta) that may be functioning in both structural and signal transduction roles. The current and prior data suggest that protein phosphorylation is central to the control of spermiation. We also present and characterize an in vitro tubule culture system that allowed functional testing of the spermiation model by pharmacologic manipulation, and yielded data consistent with the importance of protein phosphorylation in spermiation.

Fine structural observations on the process of spermiation in the holocephalan fish Hydrolagus colliei.

The process of spermiation in the ratfish Hydrolagus colliei is described and compared with that in mammals and amphibians. Spermiation in this species involves prior fluid space expansion both within the apical parts of the Sertoli cytoplasm and in the spaces between Sertoli cells and spermatids. The apical ends of Sertoli cells fragment, including the parts immediately around the spermatid acrosomes. Intercellular material between the spermatid tips and the Sertoli cells dissolves. Concurrently an opening from the seminiferous follicle into the efferent ductule is made by means of changes in cell shape and separation of Sertoli cells and efferent ductule cells that adhere to each other up to the time of sperm release.

Cytoskeletal involvement in spermiation and sperm transport

The process of spermiation and sperm transport was studied using specific inhibitors of cytoskeletal elements. Within 12–24 hr after the intratesticular injection of taxol, a compound that acts to stabilize microtubules and inhibit microtubule-related processes, an unusually large number of microtubules was seen within the body of the Sertoli cell. At the same time, transport of elements within the seminiferous epithelium was affected. At the end of stage VI of the cycle, step 19 spermatids were maintained in the deep recesses of the Sertoli cell and not transported to the rim of the seminiferous tubule lumen. At stage VIII, residual bodies remained at, or near, the rim of the tubule and were not transported to the base of the tubule. They underwent only partial degradation at this site, indicating that there may have been two phases involved in their dissolution—one autophagic and one phagocytic, but the latter did not occur since the residual bodies were not transported to Sertoli lysosomes at the base of the tubule. The observations suggest that microtubules are involved in transport processes within the seminiferous epithelium. Within 1–12 hr after the intratesticular injection of 500 μM cytochalasin D, a compound which interferes with actin-related processes, normal appearing tubulobulbar complexes were not present. The tubular portion (distal tube) of the complex did not initiate development. It was assumed that filaments (which were identified as such using NBD-phallacidin and the S-1 fragment of myosin) played an important role in the development of this portion of the complex. Cells did not eliminate cytoplasm normally, as evidenced by an enlarged cytoplasmic droplet, further emphasizing the published role for tubulobulbar complexes in cytoplasmic elimination. Although sperm were released normally from stage VIII tubules, many remained within the tubular lumen and did not traverse the duct system. Cytochalasin did not inhibit fluid secretion by the Sertoli cell, as demonstrated by efferent duct ligation, but did alter myoid cell actin cytoskeletal organization, suggesting that myoid cell contractility is primarily responsible for transport of sperm. Overall, the observations suggest that cytoskeletal activity of the Sertoli cell is important for several aspects of the spermiation process as well as sperm transport.

A comparative study of sertoli cell ectoplasmic specializations in selected non-mammalian vertebrates

Ectoplasmic specializations (ES) facing spermatids were studied in species representative of four classes of non-mammalian vertebrates (Pisces—bluegill; Amphibia—bullfrog; Reptilia—red eared turtle; Aves—domestic chicken). ES was not seen in the bluegill but was present in all other species studied. In the frog, turtle, and chicken, ES did not resemble its mammalian counterpart and could only be characterized by the presence of 6 nm filaments (presumedly actin) within the somatic cell facing the head region of elongating spermatids. ES filaments were sparse in the frog and were sometimes associated with more deeply situated endoplasmic reticulum. Turtle ES filaments were abundant and encircled the acrosomal region of the spermatid head and were delimited by fenestrated saccules of endoplasmic reticulum. In the chicken, ES filaments were prominent but less abundant than in the turtle. Six nanometer filaments of the chicken ES appeared in a tangled mass and were not associated with clearly defined endoplasmic reticulum. In the three species where ES was found, it first developed as spermatids became entrenched within the surrounding somatic cell. Neither cell elongation, nuclear elongation, or movement of the nucleus to the cell surface was synchronized with the onset of ES development. That ES development was seen concomitant with spermatid entrenchment and spermatid orientation suggested a role for ES in these processes. This hypothesis was further strengthened by observations in the fish where ES was lacking and where spermatid entrenchment within the somatic cell, did not occur. The study also supported the hypothesis that ES acts as a cytoskeletal mantle to which other cytoskeletal elements within the cell interact to affect the position of elongate spermatids within the epithelium. The dissolution of ES prior to spermiation and concomitant loss of a close relationship between cells suggests that ES is also related to somatic cell-germ cell adhesion and therefore plays an important role in the spermiation process.

A quantitative analysis of spermatogenesis in the trout, Salmo trutta fario.

The pattern of spermatogenesis was studied during an annual reprodutive cycle in adult male brown trout. The part of the testicular volume occupied by the nuclei of germ cells and of Sertoli cells, the lobular lumen and the extra-lobular space were measured. The number of germ cells was estimated by the total volume occupied by each cell divided by the individual volume of each cell. The permanent number of spermatogonia A (spermatogonia isolated or in groups of 2) was about 50 million. Each GA cell produced several spermatogenetic waves during the same reprodutive cycle. Sperm production was about 25 X 10(9)/g of testis/year, or about 6.8 X 10(7)/g of body weight/year. Males stocked separately from females, thus presumably not releasing spermatozoa, were killed in December, January and February. Since these fish contained the same amount of spermatozoa it appears that the process of spermiation initiated in December did not continue in January and February. During this period, some spermatozoa were phagocytized by macrophages (spermatophages) in the lobular lumen and by Sertoli cells.

Endocrine Control of Spermatogenesis in Teleost Fish

Very different testicular structures and spermatogenetic patterns have been found in fish of the teleost group. Two types of structures may be identified: (i) a tubular type with no lumen (in cyprinodonts); the cysts migrate from the blind end to the vas efferens during the process of spermatogenesis; (ii) a lobular type having a central lumen receiving the spermatozoa released from cysts which remain stationary along the lobule during spermatogenesis. Different spermatogenetic patterns are distinguished in salmonids and cyprinids. In the latter (carp, Cyprinus carpio, and goldfish, Carassius auratus), some germ cell types (e.g. type B spermatogonia and spermatozoa) are present throughout the year, allowing nearly continuous production of good-quality sperm. Studies of their endocrine patterns suggest that the GTH involved is controlled by external (mainly temperature but also photoperiod) and gonadal factors. The GTH stimulates androgen production and eventually controls spermatogenesis and spermiation. In salmonids, the two major events of the testicular cycle, spermatogenesis and spermiation, are temporally separated by a stage of spermatozoal "maturation" during which the spermatozoa undergo physiological changes. Sperm quality declines during the period of spermiation. The initiation of a new spermatogenetic cycle seems possible only when the spermatozoa have been eliminated from the testis, either by the normal process of spermiation or by intratesticular resorption. This also illustrates the spatial independence of spermatogenesis and spermiation. The endocrine pattern of spermatogenesis in salmonids is similar to that in carp but seems different as regards spermiation. This spermiation process includes two steps, initiation and amplification, which require different GTH levels and steroid balance. Among the steroids, 11-ketotestosterone is the major androgen found in the plasma, but its superiority over other androgens in regard to spermiation remains to be demonstrated. Environmental factors (photoperiodic changes and temperature) may act directly on the central nervous system to control gonadotropin secretion. Temperature may also directly influence the gonad, somatic or germ cells, and steroid metabolism which acts either locally on the gonad or more centrally. The regulation of spermatogenesis in fish appears to be more subtle than previously believed. Major unknowns are whether there is a second GTH and, if so, its site of action; which steroids are directly involved in the control of spermatogenesis in the lobules, and which are the target cells; and which factors regulate testicular size and which pituitary GTH secretion. Finally, the poor yield of spermiation is intriguing and requires further study, considering its practical implication in fish-farming.Key words: spermatogenesis, teleosts, salmonids, cyprinids

Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules.

Near the end of spermiogensis, the late spermatids remain attached to the superficial layer of the seminiferous epithelium for an appreciable period of time (i.e., 3 to 4 days). Ths sickle-shaped heads of the spermatids are embedded in an apical process of Sertoli cell cytoplasm which is connected to the rest of the cell by a narrow stalk. In the concavity of the head several long (2-3 mum) and very narrow (50 nm) tubular projections of the spermatid's plasma membrane invaginate the Sertoli cell cytoplasm. These tubular processes terminate by a bulbous swelling which may measure up to 1 mum in diameter. Along the process the plasma membrane of the Sertoli cell is closely apposed to the spermatid's membrane, the intracellular space being only 6-8 nm wide. In the Sertoli cytoplasm immediately surrounding the tubular portion of the structure there is an accumulation of filamentous material, while next to the bulbous extremity there are, at a shrot distance, smooth surfaced cisternae of endoplasmic reticulum. The whole structure was referred to as a tubulobulbar complex. These complexes, of which there are up to 24 per spermatid, appear as these cells complete their migration toward the apex of the Sertoli cells. They disappear just before the release of the spermatids in the lumen of the seminiferous tubule as a result of the fragmentation of the spermatid's plasma membrane followed by a resorption of the Sertoli plasma membrane. Morphological evidence suggests that the Tubulobulbar complexes serve as anchoring devices that retain the spermatids at the surface of the seminiferous epithelium while their dissolution contributes in part to the process of spermiation. Similar tubulobulbar complexes were also formed by the plasma membranes of two adjacent Sertoli cells close to the Sertoli-Sertoli tight junctions near the tubular limiting membrane.

Spermiation and sperm transport in passerine birds.

Summary. The testes and seminal sacs of thirty European goldfinches and twenty-one canaries collected from specific nests at known phases of the nest cycle were histologically examined. Although no relationship between a characteristic stage of gonadal development and a specific phase of the nest cycle could be established, the histological findings suggested a relationship between the functioning of the seminal sac and the process of spermiation. The histological results are reported and a hypothetical mechanism for spermiation and sperm transport in passerine birds is described.