- Characterization of in vivo autophagy during avian spermatogenesis1. [Journal Article]
- PSPoult Sci 2019 Jun 14
- Spermatogenesis is a complex cellular process that includes many subcellular events that are essential for the production of healthy spermatozoa. Autophagy is a physiological process that plays a sig…
Spermatogenesis is a complex cellular process that includes many subcellular events that are essential for the production of healthy spermatozoa. Autophagy is a physiological process that plays a significant role in the process of spermatogenesis; however, autophagy during avian spermatogenesis has not yet been reported. In the current study, we characterized in vivo autophagy throughout the process of domestic fowl spermatogenesis. Autophagy-specific markers, including microtubule-associated protein light chain 3 (LC3), sequestosome 1 (p62), and autophagy-related 7 (Atg7), were used to confirm the occurrence of autophagy in testicular germ cells. The protein expression of Atg7, LC3, and p62 in domestic fowl testes was confirmed by Western blotting. The immunohistochemical staining indicated a strong localization of LC3 and Atg7 within spermiogenic cells (intermediate and late spermatids) and primary spermatocytes. However, poorly expressed in cells (spermatogonia) that were located near the basement membrane. The immunofluorescence staining results showed the opposite tendency for LC3 and p62. LC3 was more strongly localized within the elongated spermatids, while p62 was strongly localized within the early spermatids. Moreover, the ultrastructural components of autophagy were revealed by transmission electron microscopy. Well-developed autophagosomes and multivesicular bodies were found to be prominent in primary spermatocytes (zygotene and pachytene) and spermiogenic cells. Furthermore, other vesicular structures, such as early endosomes and amphisomes, were also observed during spermatogenesis. The above findings collectively suggest that autophagy is active during spermatogenesis and that the level of autophagy increases from the basal to the luminal regions of the seminiferous tubules of domestic fowl testes. We propose that autophagic pathways may be involved in multiple functions to sustain spermatogenesis.
- PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis. [Journal Article]
- DDevelopment 2019 Jun 12
- Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testi…
Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2 and its RING domain, a histone writer, can ubiquitinate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitinate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.
- Effects of long-term release GnRH agonist "deslorelin" on testicular HSP expression, accessory sex glands and testicular functions in adult male rats. [Journal Article]
- TTheriogenology 2019; 134:104-111
- The objective of the present was to determine the effect of long-term release GnRH agonists "deslorelin" on suppression and restoration of testicular and accessory sex glands functions, and expressio…
The objective of the present was to determine the effect of long-term release GnRH agonists "deslorelin" on suppression and restoration of testicular and accessory sex glands functions, and expression of HSP in testes of adult male rats. A group of twenty-eight male rats and fifty-six female rats were kept for eleven months. The male rats were subdivided into treatment (n = 18; deslorelin, an analogue of GnRH, 4.7 mg, S.C; six months) and control (n = 10; untreated), and the adult female rats were introduced with either treatment or control male rats at the 2nd, 6th and 11th months post implant insertion. At 6th month of deslorelin implants insertion, six male rats from treatment and five rats from control group were sacrificed. The remaining (twelve treatment and five control) male rats were sacrificed at 11 months. The testicular dimension were measured monthly in both treatment and control rats. The blood samples were collected for testosterone and HSP70 antibody, whereas, the testes and accessory glands were isolated for histological examination at each sacrificial time. The results showed that testicular dimension were significantly lesser in treatment group until 9 months post treatment. HSP70 protein expression was negligible at 6 months in treatment group but its intensity increased in spermatids 11 months of treatment similar to control group. Significantly lower testosterone concentrations with poor semen quality, and smaller litter size were observed in treatment group. The histological picture of accessory sex glands and seminiferous tubules shown a variable integrity in treatment group than control at 6 months implant insertion. In conclusion, the subcutaneous application of 4.7 mg of the GnRH-analogue deslorelin represents a practicable, like in the female rats, method to suppress testicular, accessory sex glands functions, testicular HSP expression and fertility in male rats. Moreover, the suppressive effects of deslorelin, continued until 11th months after removal of the implant.
- The sperm associated antigen 6 interactome and its role in spermatogenesis. [Journal Article]
- RReproduction 2019 May 01
- Mammalian SPAG6, the orthologue of Chlamydomonas reinhardtii PF16, is a component of the central apparatus of the "9+2" axoneme that controls ciliary/flagellar motility, including sperm motility. Rec…
Mammalian SPAG6, the orthologue of Chlamydomonas reinhardtii PF16, is a component of the central apparatus of the "9+2" axoneme that controls ciliary/flagellar motility, including sperm motility. Recent studies revealed that SPAG6 has functions beyond its role in the central apparatus. Hence, we re-examined the role of SPAG6 in male fertility. In wild-type mice, SPAG6 was present in cytoplasmic vesicles in spermatocytes, the acrosome of round and elongating spermatids, and the manchette of elongating spermatids. Spag6-deficient testes showed abnormal spermatogenesis, with abnormalities in male germ cell morphology consistent with the multi-compartment pattern of SPAG6 localization. The armadillo repeat domain of mouse SPAG6 was used as a bait in a yeast two-hybrid screen, and several proteins with diverse functions appeared multiple times, including Snapin, SPINK2, and COPS5. Snapin has a similar localization to SPAG6 in male germ cells, and SPINK2, a key protein in acrosome biogenesis, was dramatically reduced in Spag6-deficient mice which have defective acrosomes. SPAG16L, another SPAG6 binding partner, lost its localization to the manchette in Spag6-deficient mice. Our findings demonstrate that SPAG6 is a multi-functional protein that not only regulates sperm motility, but also plays roles in spermatogenesis in multiple cellular compartments involving multiple protein partners.
- A histological, immunohistochemical and biochemical study of the effects of pomegranate peel extracts on gibberellic acid induced oxidative stress in adult rat testes. [Journal Article]
- BHBiotech Histochem 2019 May 30; :1-14
- Gibberellins are commonly used plant growth regulators that exhibit deleterious effects on various animal tissues. We investigated the histological, immunohistochemical and biochemical effects of gib…
Gibberellins are commonly used plant growth regulators that exhibit deleterious effects on various animal tissues. We investigated the histological, immunohistochemical and biochemical effects of gibberellic acid (GA3) on rat testes as well as the possible protective role of pomegranate peel extract (PPE). We used 28 adult male rats divided into control, PPE treated, GS3 treated and GA3 + PPE treated groups. Testis specimens were analyzed for superoxide dismutase (SOD) and catalase (CAT) activity, and examined histologically. We also investigated the androgen receptor using immunohistochemistry. The GA3 treated group exhibited significantly decreased SOD and CAT levels and area percent of androgen receptor. Seminiferous tubules (ST) were widely separated and the germinal epithelium was separated from the basement membrane in some tubules. Areas of vacuolation, degenerated germ cells with pyknotic nuclei and large multinucleated cells were observed. Ultrastructurally, primary spermatocytes exhibited vacuolated cytoplasm, degenerated mitochondria and hyperchromatic nuclei. Degenerated early spermatids with a ruptured or hyperchromatic nucleus were found. Spermatozoa exhibited abnormalities of the head and tail. The cytoplasm of Sertoli and Leydig cells exhibited dilated smooth endoplasmic reticulum. A significant improvement of the biochemical, histological and immunohistochemical alterations was observed in the GA3 + PPE treated group compared to the GA3 treated group.
- Tau Tubulin Kinase is required for spermatogenesis and development of motile cilia in planarian flatworms. [Journal Article]
- MBMol Biol Cell 2019 05 29; :mbcE18100663
- Cilia are microtubule-based structures that protrude from the apical surface of cells to mediate motility, transport, intracellular signaling, and environmental sensing. Tau Tubulin Kinases (TTBKs) d…
Cilia are microtubule-based structures that protrude from the apical surface of cells to mediate motility, transport, intracellular signaling, and environmental sensing. Tau Tubulin Kinases (TTBKs) destabilize microtubules by phosphorylating microtubule-associated proteins (MAPs) of the MAP2/Tau family, but also contribute to assembly of primary cilia during embryogenesis. Expression of TTBKs is enriched in testicular tissue, but their relevance to reproductive processes is unknown. We identified six TTBK homologs in the genome of the planarian Schmidtea mediterranea (Smed-TTBK-a, -b, -c, -d, -e, and -f), all of which are preferentially expressed in testes. Inhibition of TTBK paralogs by RNA-interference (RNAi) revealed a specific requirement for Smed-TTBK-d in post-meiotic regulation of spermatogenesis. Disrupting expression of Smed-TTBK-d results in loss of spermatozoa, but not spermatids. In the soma, Smed-TTBK-d RNAi impaired the function of multi-ciliated epidermal cells in propelling planarian movement, as well as the osmoregulatory function of protonephridia. Decreased density and structural defects of motile cilia were observed in the epidermis of Smed-TTBK-d(RNAi) by phase contrast, immunofluorescence, and transmission electron microscopy. Altogether, these results demonstrate that members of the TTBK family of proteins are post-meiotic regulators of sperm development and also contribute to the formation of motile cilia in the soma.
- Ultrastructure of spermiogenesis and the distribution of spermatozoal nuclear histones in the Japanese mantis shrimp, Oratosquilla oratoria (Crustacea: Stomatopoda). [Journal Article]
- JMJ Morphol 2019 May 29
- The Japanese mantis shrimp Oratosquilla oratoria (Stomatopoda; Crustacea) is one of the most economically important aquatic species of Pacific shrimp and it is distributed from Japan to the coast of …
The Japanese mantis shrimp Oratosquilla oratoria (Stomatopoda; Crustacea) is one of the most economically important aquatic species of Pacific shrimp and it is distributed from Japan to the coast of China, the Philippines, the Malay Peninsula, and the Hawaiian Islands. Early studies described certain characteristics of spermatogenesis and the sperm ultrastructure in Stomatopoda, but the composition of sperm basic nuclear proteins (SBNPs) remains completely unknown. We studied the sperm ultrastructure of O. oratoria using transmission electron microscopy and the histone composition using immunofluorescence and immunoelectron microscopy. We found that the spherical nucleus is adjacent to the electron translucent external coat, which occurs in early spermatids. The acrosomal structure begins to form at the junction of the nucleus and the external coat. At the mid-spermatid stage, part of the chromatin appears to be more electron-dense than the external coat side. The aflagellate sperm of O. oratoria, are rounded or slightly ovoid in shape and have a consistent granular nucleus, an acrosome structure of pushpin shape and a spherical vesicular body in which faintly granular material is scattered. The acrosome consists of an acrosomal vesicle, perforatorium, and subacrosomal material. The sperm contains histones H2A, H2B, H3, H4, H3.3, H2AX, and H2AZ as well as some histone modifications, that is, H3K9me3, H3K4me2, H3S10ph, H4Kac, and H2A + H4S1ph. Histones are localized not only in the nucleus of the sperm but also in other structures outside the nucleus. The results may provide new perspectives for systematic studies of crustaceans and their sperm chromatin components. These findings extend the study of the sperm structure of Stomatopoda and provide basic data to elucidate the epigenetic mechanism of fertilization.
- Histology and ultrastructure of the testis and vas deferens in Pseudochorthippus parallelus parallelus (Orthoptera, Acrididae). [Journal Article]
- MRMicrosc Res Tech 2019 May 28
- Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) is a widespread species in Europe, and also it is localized in some regions in Turkey such as Bursa, Eskişehir, Ank…
Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) is a widespread species in Europe, and also it is localized in some regions in Turkey such as Bursa, Eskişehir, Ankara, Bolu, Düzce, and Çankırı. The features of the reproductive organs such as the numbers and shapes of testes and follicles can be used as taxonomical characters. For this purpose, the ultrastructural and histological features of testis and vas deferens in P. parallelus parallelus were examined with using light microscope, scanning electron microscope, and transmission electron microscope. The mature P. parallelus parallelus has two conjugated testes produce spermatozoa. Each testis is composed of numerous testis follicles in which different stages of spermatogenesis and spermiogenesis develop. First, spermatocytes are formed by the mitosis division of the germ cells at the distal end of the follicles. Then, spermatocytes form spermatids by meiosis division in the middle region of the follicles. Finally, spermatids are differentiated to spermatozoa at the proximal region of the follicles. After maturation of the spermatozoa, sperm tails come together as the sperm bundles called as spermatodesm. Each follicle is connected to vas deferens via vas efferens to discharging spermatozoa. In spite of some differences, the testes and the vas deferens in P. parallelus parallelus are highly similar to the those of other species, especially Orthopteran species.
- Characterization and expression profiles of cyp19a1a in the schizothoracine fish Schizothorax prenanti. [Journal Article]
- TCTissue Cell 2019; 58:70-75
- Aromatase plays a central role in ovarian differentiation and development in teleosts. In the present study, we identified a cyp19a1a homologue from the ovary of Schizothorax prenanti and analysed it…
Aromatase plays a central role in ovarian differentiation and development in teleosts. In the present study, we identified a cyp19a1a homologue from the ovary of Schizothorax prenanti and analysed its expression at both the mRNA and protein levels. Cyp19a1a of S. prenanti showed high homology with that of other teleosts, especially S. kozlovi. The ovary and testis were the main sites of cyp19a1a expression in S. prenanti, and cyp19a1a transcript levels peaked in the mid-vitellogenic (MVG)-stage ovary and the mid-spermatogenic (MS)-stage testis. Signals of Cyp19a1a immunopositivity were detected in the spermatocytes and follicular cells of cortical alveolar-stage and MVG oocytes but not in spermatogonia or spermatids. Taken together, these findings indicate that Cyp19a1a may play an important role in oocyte vitellogenesis as well as spermatocyte development in S. prenanti.
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- Seminiferous tubule molecular imaging for evaluation of male fertility: Seeing is believing. [Journal Article]
- TCTissue Cell 2019; 58:24-32
- The proper assessment of male fertility is essential for diagnosing and treating male infertility. Currently, spermiogram and Johnsen testicular biopsy score counts are used to assess male fertility.…
The proper assessment of male fertility is essential for diagnosing and treating male infertility. Currently, spermiogram and Johnsen testicular biopsy score counts are used to assess male fertility. However, spermiogram is not a suitable option for non-obstructive azoospermia patients, and Johnsen testicular biopsy scores only represent localized and not the overall spermatogenesis. Whole-mount staining was a novel method for evaluating protein expression in the tissue. Thus, we explored its application in human seminiferous tubules. Testicular biopsies from 57 azoospermia patients were categorized as obstructive azoospermia (OA), maturation arrest (MA) and Sertoli-cells only syndrome (SCOS). We performed whole-mount staining of their seminiferous tubules and evaluated the spermatogonial stem cells (SSCs), differentiated spermatogonia (SG), spermatocytes (SPC) and spermatids (SD) with their respective markers (GFRA1, CD117, SYCP3, and PNA) to assess fertility. GFRA1, CD117, SYCP3, and PNA were not expressed in SCOS patients, whereas all of them were detected in OA patients. In MA patients with arrested spermatogenesis at the SPC stage, GFRA1, CD117, and SYCP3, but not PNA were expressed in the seminiferous tubules. In MA patients with arrested spermatogenesis at the spermatogonia stage, only GFRA1 was expressed in the seminiferous tubules. These results were consistent with the Johnsen testicular biopsy score counts except for one patient, where although only Sertoli cells were indicated by the score, SSCs were also detected in the whole-mounts. Collectively, whole-mount staining could be used to analyze the inherent spermatogenesis of seminiferous tubules through staining of germ cells at different stages. It offers a more accurate and promising faster method for assessing male fertility compared with traditional biopsy screening. And it could have potential value for the clinical purpose for male fertility management.