Morphometric, Histochemical, and Ultrastructural Analysis of the Reproductive System and Spermatogenic Stages of Male Blue Crab (Callinectessapidus Rathbun, 1896)

The blue crab, Callinectes sapidus Rathbun, 1896, is considered to be a luxury meal, especially in touristic cities. It contains more than 20 types of amino acids and provides all the needed amounts for human growth. This study describes the morphology and complex differentiation in the gonadosomatic index, morphological and ultrastructure features of the reproductive system, spermatogenesis, and spermatophores structure; this is due to the need to maintain natural and fishing stocks. Mature adult male crabs (carapace length 59 ± 7.12; width 126 ± 18.8 mm) were obtained from Abu-Qir Bay from November 2018 to October 2019 and transported alive in seawater to the laboratory. The reproductive system was dissected and weighed to the nearest 0.001g using the electronic balance, and the gonadosomatic index was subsequently calculated. The morphological analysis showed the developing testes with highly compacted seminiferous tubules. Using Periodic acid–Schiff stain, the spermatophore appeared with a zigzag-shaped wall that indicates its carbohydrate constituents. Each Spermatophore consisted of an inner spermatozoal mass embedded in a matrix, whose main components were secretions 1 and 2, and an outer thin acellular layer composed of secretions 3 and 4 from the anterior vas deferens (AVD). Secretions 5 and 6 (S5 and S6) also appeared with carbohydrate constituents using Mallory triple stain. The transverse section of the middle vas deferens (MVD) showed spermatophores with rod-shaped secretion S6 and granular secretions 7 (S7), forming a complex matrix between spermatophores. The secretion found in the MVD was granular, strongly acidophilic, and secreted by its highly columnar epithelium (S7). The ultrastructure showed that the testes were surrounded by a monolayer of myoid cells with an elongated nucleus, which also contained the following stages: spermatogonia, primary spermatocyte, secondary spermatocytes, and spermatids as well as spermatozoa. On the other hand, scanning electron microscope studies for fully formed spermatophore taken from the middle part of the vas deferens indicated that it is ellipsoidal in its outline with terminal stalk. Furthermore, the spermatophore was surrounded by a thick capsule of non-cellular substances and contained mature spermatozoa.

The ameliorative effect of ellagic acid on di-(2-ethylhexyl) phthalate-induced testicular structural alterations, oxidative stress, inflammation and sperm damages in adult mice

Background Phthalates such as di (2-ethylhexyl) phthalate (DEHP) are well known exogenous substances, disrupting reproductive system function and structure. The current research demonstrated the effect of ellagic acid (EA) on DEHP-induced testicular injury in mice. Methods Thirty-five healthy adult male mice were randomly divided to five groups; normal saline receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) receiving group, DEHP (2 g/kg/day, dissolved in corn oil, p.o.) and EA receiving groups (25, 50 and 100 mg/kg/day, p.o.). Treatment duration of animals was 14 days. Body and testes weights and sperm characteristics and histological changes of testes were evaluated. Serum testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were analyzed. In the testicular tissue, oxidative/nitrosative stress markers and inflammatory cytokine levels were measured. Results Ellagic acid significantly reduced DEHP-induced reduction of body and testes weights. The DEHP-induced reduction of spermatogonia, primary spermatocyte and sertoli cells numbers as well as reduction of sperm vitality and progressive motility were reversed by EA. Furthermore, EA inhibited DEHP-induced alterations in serum hormone levels. These effects were associated with the reduction of DEHP-induced increased level of oxidative stress and inflammatory responses. Conclusions Ellagic acid considerably inhibits testicular toxicity of DEHP through reducing oxidative/nitrosative stress and inflammatory responses. Our data suggest that EA may be considered as a promising agent to inhibit male reproductive toxicity induced by endocrine disrupting chemicals such as DEHP.

IGSF11 is required for pericentric heterochromatin dissociation during meiotic diplotene

Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic and germ cells are required for this regulation. Our results show that deficiency for adhesion molecule IGSF11, which is expressed in both Sertoli cells and germ cells, leads to male infertility in mice. Combining a new meiotic fluorescent reporter system with testicular cell transplantation, we demonstrated that IGSF11 is required in both somatic cells and spermatogenic cells for primary spermatocyte development. In the absence of IGSF11, spermatocytes proceed through pachytene, but the pericentric heterochromatin of nonhomologous chromosomes remains inappropriately clustered from late pachytene onward, resulting in undissolved interchromosomal interactions. Hi-C analysis reveals elevated levels of interchromosomal interactions occurring mostly at the chromosome ends. Collectively, our data elucidates that IGSF11 in somatic cells and germ cells is required for pericentric heterochromatin dissociation during diplotene in mouse primary spermatocytes.

The Novel Key Genes of Non-obstructive Azoospermia Affect Spermatogenesis: Transcriptomic Analysis Based on RNA-Seq and scRNA-Seq Data

Non-obstructive azoospermia (NOA) is one of the most important causes of male infertility. It is mainly characterized by the absence of sperm in semen repeatedly or the number of sperm is small and not fully developed. At present, its pathogenesis remains largely unknown. The goal of this study is to identify hub genes that might affect biomarkers related to spermatogenesis. Using the clinically significant transcriptome and single-cell sequencing data sets on the Gene Expression Omnibus (GEO) database, we identified candidate hub genes related to spermatogenesis. Based on them, we performed Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analyses, protein-protein interaction (PPI) network analysis, principal component analysis (PCA), cell cluster analysis, and pseudo-chronological analysis. We identified a total of 430 differentially expressed genes, of which three have not been reported related to spermatogenesis (C22orf23, TSACC, and TTC25), and the expression of these three hub genes was different in each type of sperm cells. The results of the pseudo-chronological analysis of the three hub genes indicated that TTC25 was in a low expression state during the whole process of sperm development, while the expression of C22orf23 had two fluctuations in the differentiating spermatogonia and late primary spermatocyte stages, and TSACC showed an upward trend from the spermatogonial stem cell stage to the spermatogenesis stage. Our research found that the three hub genes were different in the trajectory of sperm development, indicating that they might play important roles in different sperm cells. This result is of great significance for revealing the pathogenic mechanism of NOA and further research.

Histological, histochemical, immunohistochemical and ultrastructural characterization of the testes of the dove

Summary Avian testes have been used in the study of germ cell transfer, importantly for understanding the preservation and control of birds. For this purpose, we use light microscopy, electron microscopy and immunohistochemistry to understand the reproductive efficiency of dove testes. The tunica albuginea was thin and septula testes were not observed. The testicular parenchyma was formed mainly of closely packed convoluted seminiferous tubules with little interstitial area. Three types of spermatogonia were distinguished. The primary spermatocyte appeared as the largest spermatogenic cell and was identified at different stages of meiosis. Different morphological stages of the spermatid were categorized. Various cellular associations were described within the seminiferous epithelium. The cytoplasm of Sertoli cells was pale and ill defined due to its close relationship to the germinal epithelium. The spermatid attached to the luminal border of Sertoli cells and germ cells were closely associated. A single layer of myoid cells surrounded the seminiferous tubule. Testicular telocytes of doves were located in the peritubular region and near the blood vessels. Telopods appeared as long cytoplasmic processes arising from the cell body. Leydig cells were distributed singly or in small groups and cords. The intensity of androgen receptor (AR) immunostaining in the testes of the dove was established for the first time and is described in this paper.

Effects Of Short And Long-Term Administration Of Alfalfa On Testicular Histomorphometry In Rats

Alfalfa (Medicago sativa) is a plant with phytoestrogenic properties, which has been used as a major part of diets in husbandry. Since there are controversial reports related to the effects of alfalfa consumption on animal fertility, its effects on rat testicular tissue were assessed in the present study. Control (n=15) and alfalfa (n=15) groups were fed with ordinary rat chow and ordinary rat chow plus alfalfa, respectively. Testicles were removed after 30, 45, and 60 days of consumption, and tissue sections were prepared to assess histomorphometric changes related to alfalfa consumption. Based on the results, there was no significant difference in length, width, and volume of testes of treated rats to control in all groups. But the number of testicular spermatogonia cells, primary spermatocyte cells, primary spermatid cells, testicular spermatozoid cells and Leydig cells significantly or insignificantly increased in rats that received alfalfa for 30 days but all of these parameters insignificantly decreased in rats that received alfalfa for 60 days. The cause of these changes may be due to estrogenic or anti-estrogenic, antioxidant and endocrine effects of alfalfa. Conclusion: Consumption of alfalfa for short time had only a transient positive effects on testicular tissues but use of alfalfa for 60 days had little destructive effects on testicular tissue in rats. So longer durations of time could be suggested for further research on the effects of alfalfa on rat’s reproduction index.

EFEK PEMBERIAN L-ARGININ TERHADAP GAMBARAN HISTOLOGI JUMLAH SPERMATOSIT PRIMER PADA MENCIT (Mus musculus) SETELAH TERPAPAR SUHU PANAS

The purpose of this study was to know the effect of L-arginine on the histology of primary spermatocyte count in mice (Mus musculus) after high temperature exposed. The subjects of this study were 20 adult male mice, 8 weeks old with an body weight range from 20-40 grams. This research conducted by using Complete Randomized Design (RAL) with 4 treatments and 5 replications. The treatments consisted of, P0- = treatment with 1ml of aquabidest without high temperature exposed, P0 + = treatment with 1ml of aquabidest after high temperature exposed for 1hour per-day for 35 days, P1 = 1.3mg / day L-arginine dissolved in 1ml aquabidest and given orally after high temperature exposed for 1hour per-day for 35 days and P2 = 2.6mg / day L-arginine dissolved in 1ml aquabidest given orally after high temperature exposed for 1hour per-day for 35 days. Observations done by making histologic preparations of testicular organs and then calculated the total number of primary spermatocyte per treatment. The data of primary spermatocyte calculated and analyzed by using Analysis of Variance (ANOVA), followed by Duncan test. The result from data analysis showed that there was a significant difference (p<0.05) between P1 and P2 with control,between P1 and P2 the analysis did not show any significant difference (p> 0.05).

Spermatogenesis Mencit (Mus musculus, L.) Pasca Pemberian Ekstrak Brotowali (Tinospora crispa, L.)

ABSTRAKBrotowali (Tinospora crispa, L.) merupakan tumbuhan yang mengandung senyawa yang tergolong dalam kelompok antifertilitas diantaranya golongan glikosida, alkaloid, flavonoid, saponin dan tanin. Kelompok senyawa tersebut dapat memberikan efek sitotoksik pada reproduksi jantan dengan mengganggu metabolisme sel germinal dan sel spermatogenik. Tujuan penelitian ini untuk mengetahui spermatogenesis mencit pasca diberikan ekstrak brotowali. Sebanyak 20 ekor mencit jantan berat 30-40 gr, berumur 2-3 bulan dibagi 5 kelompok perlakuan yaitu; K1 kontrol positif (aquades), K2 kontrol negatif (Na CMC 0,5% ), K3 (0,05 g/g bb), K4 (0,06 g/g bb), dan K5 (0,07 g/g bb) ekstrak brotowali. Ekstrak diberikan secara oral setiap hari selama 34 hari. Pada hari ke-35 mencit dikorbankan dengan kloroform selanjutnya pengambilan organ testis untuk dibuat preparat mikroanatomi. Data dianalisis dengan ANOVA dan uji BNT (α=0,05%). Hasil penelitian menunjukkan bahwa pemberian ekstrak tumbuhan brotowali secara signifikan mengganggu proses spermatogenesis mencit dengan menurunkan jumlah sel spermatogonium, spermatosit primer, sel spermatid dan kepadatan spermatozoa. Penurunan jumlah sel-sel spermatogenik dalam penelitian ini terbanyak pada perlakuan K5 untuk semua kelompok, dengan jumlah spermatogonium (28,5), dengan jumlah spermatosit primer (33,3), dengan jumlah spermatid (69,4) dan kepadatan spermatozoa (1,19). Kata kunci : Ekstrak, Tinospora crispa, L., Spermatogenesis, Mencit ABSTRACTBrotowali (Tinospora crispa, L.) is a plant that contains compounds belonging to the antifertility group including glycosides, alkaloids, flavonoids, saponins and tannins. This group of compounds can provide cytotoxic effects on male reproduction by disrupting germinal cell metabolism and spermatogenic cellsThe aim of this study was to know the effect of extracts Tinospora crispa, L. on spermatogenesis of mice (Mus musculus, L.). The male mice were used 20 with weight 30-40 g, aged 2-3 months, and were treated in 5 groups i.e. K1 : positive control (aquadest), K2 : negative control (Na CMC 0.5%), K3 (0.05 g/g bb), K4 : (0.06 g/g bb), and K5 (0.07 g/g bb) extracts of brotowali. The extracts were given orally during 34day. At the 35th the weight of mice werw counted and then sacrificed with chloroform, and the testis were collected to make microanatomy. The data were analyzed with ANOVA (Analysis of Varience) and t-Test (α = 0,05%). The results showed that the treatments with brotowali extracts obstructed spermatogenesis process and reduced the amount of spermatogonium cells, primary spermatocyt, spermatide cells and density of spermatozoa with increasing dosage of brotowali extracts. The decreasing of spermatogenic cells were observed in dosage of K5 with spermatogonium cells (28,5), the amount primary spermatocyte (33,3), the amount of spermatide (69,4) and the density of spermatozoa (1,19).Key word : Extracts, Tinospora crispa, L., Spermatogenesis, Mice

Extraordinary centromeres: differences in the meiotic chromosomes of two rock lizards speciesDarevskia portschinskiiandDarevskia raddei

According to the synthesis of 30 years of multidisciplinary studies, parthenogenetic species of rock lizards of genusDarevskiawere formed as a result of different combination patterns of interspecific hybridization of the four bisexual parental species:Darevskia raddei,D. mixta,D. valentini, andD. portschinskii. In particular,D. portschinskiiandD. raddeiare considered as the parental species for the parthenogenetic speciesD. rostombekowi. Here for the first time, we present the result of comparative immunocytochemical study of primary spermatocyte nuclei spreads from the leptotene to diplotene stages of meiotic prophase I in two species:D. portschinskiiandD. raddei. We observed similar chromosome lengths for both synaptonemal complex (SC) karyotypes as well as a similar number of crossing over sites. However, unexpected differences in the number and distribution of anti-centromere antibody (ACA) foci were detected in the SC structure of bivalents of the two species. In all examinedD. portschinskiispermatocyte nuclei, one immunostained centromere focus was detected per SC bivalent. In contrast, in almost every studiedD. raddeinuclei we identified three to nine SCs with additional immunostained ACA foci per SC bivalent. Thus, the obtained results allow us to identify species-specific karyotype features, previously not been detected using conventional mitotic chromosome analysis. Presumably the additional centromere foci are result of epigenetic chromatin modifications. We assume that this characteristic of theD. raddeikaryotype could represent useful marker for the future studies of parthenogenetic species hybrid karyotypes related toD. raddei.

Extraordinary centromeres: differences in the meiotic chromosomes of two rock lizards species Darevskia portschinskii and Darevskia raddei

According to the synthesis of 30 years of multidisciplinary studies parthenogenetic species of rock lizards of genus Darevskia were formed as a result of different combination patterns of interspecific hybridization of the four bisexual parental species: D. raddei, D. mixta, D. valentini, and D. portschinskii. In particular D. portschinskii and D. raddei are considered as the parental species for the parthenogenetic species D. rostombekowi. Here for the first time we present the result of comparative immunocytochemical study of primary spermatocyte nuclei spreads from the leptotene to diplotene stages of meiotic prophase I in two species: D. portschinskii and D. raddei. We observed similar chromosome lengths for both synaptonemal complex (SC) karyotypes as well as similar number of crossing over sites. However, unexpected differences in the number and distribution of anti-centromere antibody (ACA) foci were detected in the SC structure of bivalents of the two species. In all examined D. portschinskii spermatocyte nuclei, one immunostained centromere focus was detected per SC bivalent. In contrast, in almost every studied D. raddei nuclei we identified three – nine SCs with additional immunostained ACA foci per SC bivalent. Thus, the obtained results allow us to identify species-specific karyotype features, previously not been detected using conventional mitotic chromosome analysis. Presumably the additional centromere foci are result of epigenetic chromatin modifications. We assume that this characteristic of the D. raddei karyotype could represent useful marker for the future studies of parthenogenetic species hybrid karyotypes related to D. raddei.