Identification of SRY‐box 30 as an age‐related essential gatekeeper for male germ‐cell meiosis and differentiation

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY‐box 30 (Sox30) is an age‐related and essential regulator of meiosis in the postnatal testis. Sox30‐null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ‐cell differentiation and irregular Leydig cell proliferation. In aged Sox30‐null mice, the observed testicular impairments were more severe. Furthermore, the germ‐cell arrest occurred at the stage of meiotic zygotene spermatocytes, which is strongly associated with critical regulators of meiosis (such as Cyp26b1, Stra8 and Rec8) and sex differentiation (such as Rspo1, Foxl2, Sox9, Wnt4 and Ctnnb1). Mechanistically, Sox30 can activate Stra8 and Rec8, and inhibit Cyp26b1 and Ctnnb1 by direct binding to their promoters. A different Sox30 domain required for regulating the activity of these gene promoters, providing a “fail‐safe” mechanism for Sox30 to facilitate germ‐cell differentiation. Indeed, retinoic acid levels were reduced owing to increased degradation following the elevation of Cyp26b1 in Sox30‐null testes. Re‐expression of Sox30 in Sox30‐null mice successfully restored germ‐cell meiosis, differentiation and Leydig cell proliferation. Moreover, the restoration of actual fertility appeared to improve over time. Consistently, Rec8 and Stra8 were reactivated, and Cyp26b1 and Ctnnb1 were reinhibited in the restored testes. In summary, Sox30 is necessary, sufficient and age‐associated for germ‐cell meiosis and differentiation in testes by direct regulating critical regulators. This study advances our understanding of the regulation of germ‐cell meiosis and differentiation in the postnatal testis.     

SPERMATOGENESIS IN A TRICHUROID NEMATODE,TRICHURIS MURIS. II. FINE STRUCTURE OF PRIMARY SPERMATOCYTE AND FIRST MEIOTIC DIVISION

Ultrastructural observations indicate that the primary spermatocyte of Trichuris muris is larger than the spermatogonial stage with an increased cytoplasm to nucleus ratio. The cytoplasm contains an extensive reticular system, mitochondria, numerous free ribosomes and prominent Golgi complexes which may contribute to the formation of a sub-surface, vesicular complex. Although only two spermatocytes were seen to be linked by a cytoplasmic bridge it is suggested that the number of conjoined cells is probably greater. The rearrangement of mitochondria in a ring around the nucleus and the indentation and vesiculation of the nuclear envelope preceeded its disappearance and indicated the onset of meiosis. Centrioles were frequently resolved at this stage. They were composed of nine peripheral doublets surrounded by a dense pericentriolar sheath. Three dense chromatin areas indicative of haploid chromosomes were present in later meiotic stages. Each chromosome was surrounded by a number of mitochondria and there was a clear separation of the chromosome-mitochondrial clusters from the remainder of the cytoplasm. This was particularly evident at telophase when two daughter cells were partially separated by membrane infoldings. This reflects incomplete cytokinesis in the dividing spermatocyte of T. muris and is similar to that described in other trichuroid species. A close association with processes of the non-germinal, sustentacular cells was noted throughout the spermatocyte stage.     

Time course of meiotic progression after transferring primary spermatocyte into ooplasm at different stages

This study attempted to investigate the time course of meiotic progression after transferring primary spermatocyte (PS) into ooplasm at different maturing stages. In present experiments, PSs were introduced into maturing ooplasts or oocytes by electrofusion. Higher fusion rate was obtained by phytohemagglutinin (PHA) agglutination than by perivitelline space (PVS) insertion. When the ooplasms prepared at 0, 2, 5, and 8.5 hr of in vitro maturation (IVM) were used as recipients and PSs were used as donors, the reconstructed cells extruded the first polar body (PB1) approximately 8.5, 7, 5.5, and 3 hr after electrofusion, respectively. Especially, when ooplasm cultured for 8.5 hr in vitro after GV removal was fused with PS, the PB1 was emitted 7-11 hr after electrofusion. Additionally, the PB1 extrusions of GV and pro-MI oocytes fertilized with PSs were 2.5 hr earlier than control oocytes. The results suggest that (1) PSs undergo the first meiosis in different time courses when introduced into ooplasm at different maturing stages; (2) GV material plays an important role in determining the timing of PB1 extrusion; and (3) first meiotic division of GV and pro-MI oocytes can be accelerated by introducing PS.     

Relevance of caspase activity during apoptosis in pubertal rat spermatogenesis

Caspases are a family of cysteine-proteases, activated upon several different stimuli, which execute apoptosis in many cell death models. Previous work of our group has shown rats have the highest rate of apoptosis during the first wave of spermatogenesis (between 20 and 25 days after birth), as evaluated by TUNEL and caspase activity. However, the hierarchical order of caspase activation and the relevance of each caspase during germ cell apoptosis are not clear. Thus, the goal of this work is to take a pharmacological approach to dissect the apoptosis pathway of caspase activation. Results showed that intratesticular injection of a caspase-8 inhibitor (z-IETD-fmk), or a pan-caspase inhibitor (z-VAD- fmk), significantly decreased the cleavage of p115 and PARP, two endogenous substrates of caspases, in 22-day-old rats. Additionally, these inhibitors promoted a significant reduction in the number of apoptotic germ cells. On the other hand, intratesticular injection of two different inhibitors of the intrinsic pathway (z-LEHD-fmk and minocycline) did not have any effect upon caspase substrates cleavage (p115 and PARP) or the number of apoptotic germ cells. Therefore, we conclude that the extrinsic pathway of apoptosis plays an important role in physiological germ cell apoptosis during the first round of spermatogenesis in the rat.     

Whole body exposure of rats to microwaves emitted from a cell phone does not affect the testes

The objective of this study was to investigate the effects of radiofrequency radiation emitted from cellular phones on the lipid composition, malondialdehyde concentration, p53 immune reactivity, sperm count, morphology, histological structure of testes, and on rectal temperature of rats exposed to microwave radiation emitted from cellular phones. Sixteen Spraque-Dawley rats were separated into two groups of eight, sham exposed (control) and experimental. The rats were confined in plexiglas cages specially designed for this study, and cellular phones were placed 0.5 cm under the cages. For the experimental group, cellular phones were activated 20 min per day (7 days a week) for 1 month. For the control group, the cellular phones were placed beneath the cages for 20 min a day, but the phones were turned off. Rectal temperatures were measured weekly. For 250 mW radiated power, the whole body average SAR (rms) is 0.52 W/kg and 1 g averaged peak SAR (rms) is 3.13 W/kg. The Mann-Whitney U-test was used for statistical comparisons of groups. No statistically significant alteration in any of the endpoints was noted. This study found no evidence suggesting an adverse effect of cell phone exposure on measures of testicular function or structure.     

H2AX regulates meiotic telomere clustering

The histone H2A variant H2AX is phosphorylated in response to DNA double-strand breaks originating from diverse origins, including dysfunctional telomeres. Here, we show that normal mitotic telomere maintenance does not require H2AX. Moreover, H2AX is dispensable for the chromosome fusions arising from either critically shortened or deprotected telomeres. However, H2AX has an essential role in controlling the proper topological distribution of telomeres during meiotic prophase I. Our results suggest that H2AX is a downstream effector of the ataxia telangiectasia-mutated kinase in controlling telomere movement during meiosis.     

MORPHOGENESIS OF THE AXONEME IN THE SPERMATOCYTE OF COCCINELLA SEPTEMPUNCTATA LINNAEUS

Abstract  Using cell whole mount preparation, early morphogenesis and ultrastructure of the axoneme of Coccinella septempunctata L. spermatocyte were investigated by transmission electron microscope. During spermatogenesis two pairs of basal body-axoneme complexes originated from centrioles are found in the spermatocyte and they are separated completely from each other at interkinesis. The centriolar adjunct begins to generate while a basal body-axoneme complex is attached to the nuclear envelope of a spermatid nucleus, and it, on the proximal end of a growing axoneme, reaches a maximum before chromatin condensation. The growing axoneme is accompanied by the condensable nucleus elongation. The early axoneme of a basal body-axoneme complex consists nine doublets with only inner and outer dynein arms, no central microtubules.     

雄激素受体在大鼠睾丸中转录模式的分析

目的：研究Ar（雄激素受体）基因在大鼠睾丸组织中的转录模式。方法：取刚出生的雄性Wistar大鼠,于出生2~65日的不同时间点,脊椎脱臼处死3只不同窝别的幼鼠,提取睾丸组织总mRNA;将mRNA反转录成cDNA,随后采用Real-time PCR方法检测大鼠睾丸组织中Ar mRNA的转录情况。结果：Ar mRNA表达在出生后第2天开始缓慢上升到第16天达到最高值,第16天到第30天迅速下降,第31天出现一个小高峰后到第65天持续缓慢下降。结论：Ar基因在大鼠早期发育睾丸组织中表达量逐渐升高,可能与精原干细胞的增殖及初级精母细胞的发生相关。     

Aquaporins in gametogenesis of vertebrate animals

A review of the data on the presence, localization, and supposed role of aquaporin water channels in oocytes of Xenopus laevis, oogenesis and maturation of teleosts Sparus auratus and Oncorhynchus mykiss, oogenesis and oocyte maturation of rats and mice, and spermatogenesis of several mammalians.