Classification of the spermatogenic cycle,seasonal changes of seminiferous tubule morphology and estimation of the breeding season of the large Japanese field mouse (Apodemus speciosus) in Toyama and Aomori prefectures, Japan

The large Japanese field mouse, Apodemus speciosus, is a potential indicator of environmental stress, but this function has not been confirmed by histological studies. Since environmental stress affects the reproductive function of mice, we determined the reproductive characteristics of this species at two locations: Toyama (36°35ʹN, 137°24ʹE) and Aomori (40°35ʹN, 140°57ʹE). Mice were captured during May–November (n=119) and July–November (n=146) at these locations, respectively. We classified the breeding season from the numbers of pregnant females and young, in addition to the spermatogenic cycle and seasonal changes in seminiferous tubule morphology of males. Testicular weight was measured, and seminiferous tubule morphology was examined histologically. Fourteen stages were found in the seminiferous epithelium cycle based on acrosome formation and spermatid head morphology. At both locations, the breeding season peaked from late summer to early autumn and possibly in spring. Spermatogenic activity was classified into 4 periods from June to November: resting around June and October–November; resumptive around July; active around August; and degenerative around September. During the resting period, the seminiferous tubules consisted of Sertoli cells, spermatogonia and spermatocytes. Spermatogenesis began during the resumptive period, and spermatids were observed. During the active period, active spermatogenesis and a broad lumen were observed. During the degenerative period, spermatogenesis ended, and Sertoli cells, spermatogonia, spermatocytes and degenerating exfoliated round spermatids were observed. This study provides scientific information about the testicular histopathological evaluations of the large Japanese field mouse for its use as an index species of environmental pollution.     

达乌尔黄鼠精子形成和细胞色素芳香化酶免疫位置的季节变化

用组织学和免疫组织化学方法调查达乌尔黄鼠精子形成季节性变化和细胞色素芳香化酶(P450 arom)在精巢和附睾中的免疫位置。黄鼠繁殖期与非繁殖期精巢大小、重量、生精小管直径存在显著差异;黄鼠繁殖期精巢中存在从精原细胞到有尾精子各期生殖细胞,非繁殖期精巢中只存在精原细胞和初级精母细胞。另外,繁殖期黄鼠附睾管中存在大量有尾精子,而非繁殖期附睾中未见精子存在。繁殖期P450 arom在黄鼠精巢的间质细胞、支持细胞、精子细胞和附睾头部输出小管上皮细胞都有发现,而在非繁殖期没有发现它的活力。这些结果表明达乌尔黄鼠精子形成、成熟是伴随着精巢复发和退行呈现显著季节性变化,雌激素在精子形成和成熟过程中起着重要的生理性作用。     

Postnatal testicular development and actin appearance in the seminiferous epithelium of the Habu,Trimeresurus flavoviridis

The postnatal testicular development and actin distribution in the seminiferous epithelium were examined by light microscopy, using the testes of the Habu (Trimeresurus flavoviridis; snake) from 0-year-old to 3-year-old. At 0-year-old (about 1 month after birth), the testis was quite small in size, and the seminiferous epithelium was composed of only Sertoli cells and large spermatogonia. Actin immunoreactivity was observed in the peritubular myoid cells, but could not be detected in the seminiferous epithelium. At 1-year-old (about 10 months after birth), the testicular size increased to a great degree. In the seminiferous epithelium, spermatocytes newly appeared. Actin could still not be detected in the seminiferous epithelium. At 2-year-old (about 1 year and 10 months after birth), the testes continued to develop in size. In the seminiferous epithelium, elongate spermatids and round spermatids were frequently seen, in addition to Sertoli cells, spermatogonia and spermatocytes. Thus, active spermatogenesis was clearly recognized at this age. Moreover, the actin distribution in the seminiferous epithelium was observed at the site between Sertoli cells and spermatids, as well as that at adult stage. The immunoreactivity of actin in the peritubular myoid cells gradually increased from 0-year-old to 2-year-old. Conclusively, it seems likely that spermatogenesis in the Habu initiates at 2-year-old, accompanying with the appearance of actin in the seminiferous epithelium.     

Vitamin A Deprivation Affects the Progression of the Spermatogenic Wave and Initial Formation of the Blood-testis Barrier,Resulting in Irreversible Testicular Degeneration in Mice

The blood testis-barrier (BTB) is essential for maintaining homeostasis in the seminiferous epithelium. Although many studies have reported that vitamin A (VA) is required for the maintenance of spermatogenesis, the relationships between the BTB, spermatogenesis and VA have not been elucidated. In this study, we analyzed BTB assembly and spermatogenesis in the testes of mice fed the VA-deficient (VAD) diet from the prepubertal period to adulthood. During the prepubertal period, no changes were observed in the initiation and progression of the first spermatogenic wave in mice fed the VAD diet. However, the numbers of preleptotene/leptotene spermatocytes derived from the second spermatogenic wave onwards were decreased, and initial BTB formation was also delayed, as evidenced by the decreased expression of mRNAs encoding BTB components and VA signaling molecules. From 60 days postpartum, mice fed the VAD diet exhibited apoptosis of germ cells, arrest of meiosis, disruption of the BTB, and dramatically decreased testis size. Furthermore, vacuolization and calcification were observed in the seminiferous epithelium of adult mice fed the VAD diet. Re-initiation of spermatogenesis by VA replenishment in adult mice fed the VAD diet rescued BTB assembly after when the second spermatogenic wave initiated from the arrested spermatogonia reached the preleptotene/leptotene spermatocytes. These results suggested that BTB integrity was regulated by VA metabolism with meiotic progression and that the impermeable BTB was required for persistent spermatogenesis rather than meiotic initiation. In conclusion, consumption of the VAD diet led to critical defects in spermatogenesis progression and altered the dynamics of BTB assembly.     

Analysis of various antigens in golden hamster testis by monoclonal antibodies.

A total of 38 hybridomas producing monoclonal antibodies (mAbs) was established by immunizing BALB/c mice with extracts of the golden hamster testis. Six mAbs stained the acrosome of developing spermatids by immunofluorescence. Two mAbs (1A11 and 4D8) reacted with spermatid components other than acrosome. The mAbs 1C9 and 4D3 recognized a 103 kilodalton (kDa) protein on immunoblots, and were reactive to spermatocytes and early spermatids, but not to late spermatids and spermatozoa. This finding suggests that the protein functions for meiosis or early spermiogenesis. Four mAbs (3G2, 2E5, 2G3, and 3F10) stained all stages of spermatogenic cells. The remaining 24 mAbs showed a positive reaction to the basement membrane of the seminiferous tubule. Two of them, 3D6 and 3E5, recognized approximately 150 kDa major proteins, indicating that the antigen is an extracellular matrix.     

Evaluation of testicular toxicity and sperm morphology in rats treated with methyl methanesulphonate (MMS)

Methyl methanesulphonate (MMS), a potent alkylating agent and testicular toxicant, was orally administered to rats for 5 days at 40 mg/kg. During the recovery period of up to 5 weeks, males were evaluated for testicular toxicity and sperm morphology. The 5-week recovery period were designated as follows: Day 1 (the day after final treatment); Week 1, Week 2, Week 3, Week 4 and Week 5 (1, 2, 3, 4 and 5 weeks after final treatment). Morphologically abnormal sperm increased beginning in Week 3, peaked in Week 4 and declined slightly in Week 5. Histopathological examinations indicated retention of step 19 spermatids at stage IX from Day 1 through Week 3. Quantitative evaluation of spermatogenic cells indicated a decrease in the number of late pachytene spermatocytes and early spermatids on Day 1. TUNEL examination showed a significantly high frequency of apoptosis in the meiosis cells in Week 1. In the present study, genetic damage induced by treatment with MMS affected spermatogenesis and a wide variety of spermatogenic cells in the testis. Apoptosis in the course of meiosis seemed to be involved in the elimination process of genetically insulted germ cells, and this process seems to play an important role in eliminating and/or decreasing the germ cells with retention of spermatids and the potential to express morphologically abnormal spermatozoa.     

Seasonal changes in spermatogenesis and testicular steroidogenesis in wild male raccoon dogs (Nyctereutes procynoides)

Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3 betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.     

Immunohistochemical study of osteopontin in boar testis

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.     

Sexing of Dog Sperm by Fluorescence In Situ Hybridization

Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.     

Lectin-binding patterns in the spermatogenic cells of the shiba goat testis.

Lectin-binding patterns in the testis of the sexually mature goat were investigated by light and transmission electron microscopy. Dolichos biflorus agglutinin (DBA) and Griffonia simplicifolia agglutinin-I (GS-I) were negative in the seminiferous epithelium, but soybean (Glycine max) agglutinin (SBA), Griffonia simplicifolia agglutinin-II (GS-II) and peanut (Arachis hypogaea) agglutinin (PNA) were positive in the acrosomal vesicle of the Golgi-phase spermatids and in the acrosome of the cap-, acrosome- and maturation-phase spermatids. In addition, PNA was positive also in the plasma membrane and cytoplasm of the spermatogenic cells from the late pachytene spermatocytes to the maturation-phase spermatids. This finding indicates that glycoconjugates containing D-galactose residue appear at this period. Therefore, PNA may be a useful marker of developing spermatogenic cells. Electron microscopically, the positive reactions of SBA, GS-II and PNA were demonstrated in the central dense area (acrosomal granule) of the cap-phase spermatid acrosome, but not in the peripheral low-dense area. These results indicate that the lectin-bindings in the goat testis not only are common features, but also have patterns somewhat different from those in other mammals in such a way that GS-II can be detected in the acrosomal region of all phases of spermatids and that PNA is positive in the late spermatid acrosome.     

A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.     

Relationship of absolute numbers of Sertoli cells to testicular size and spermatogenesis in young beef bulls

Testes were obtained from 34 Hereford or Angus bulls at about 1.5 yr of age and were used to investigate the relationship between the absolute number of Sertoli cells vs testicular size and daily spermatozoal production (DSP). Quantitative determination of DSP was based upon enumeration of elongated spermatids in testicular homogenates. The ratio of step 8 spermatids to Sertoli cells (S:SC) was established by direct counts of these cells in each of 20 round stage VIII seminiferous tubular cross sections for each bull. The number of Sertoli cells per paired testes was calculated as (total spermatids divided by S:SC)/.394, where total spermatids equalled the number of homogenization-resistant spermatids. The factor of .394 adjusted for the fact that the latter cells are present for only 39.4% of the spermatogenic cycle. All data were subjected to simple linear and second-order regression analyses. A positive linear relationship (P less than .005) was found between testicular weight (Y, in grams) and the absolute number of Sertoli cells per paired testes (X, in billions), which was characterized by the equation Y = 315.2 + 10.74X and a coefficient of correlation (r) of .56 (P less than .01). A similar relationship was observed between DSP (Y, in billions) and Sertoli cell numbers (X, in billions). This was characterized by the equation Y = 1.36 + .222X (P less than .005) and a coefficient of correlation of .70 (P less than .01). Daily sperm production was unrelated to the S:SC ratio (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of the Hinny (Equus asinus × Equus caballus) Seminiferous Epithelium

The gonads and the germinative cells of 3 male hinnies were studied with light and transmission electron microscopy with the aim to observe the development of germ cells and verify the morphological modifications due to the hybridization. The hinny seminiferous epithelium presented Sertoli cells and spermatogonia with normal features and anomalous spermatocytes I. The other cells from the spermatogenic sequence were not seen. Most of the alterations began to occur in the cytes I, which presented nuclear vacuolization and deposits of amorphous material between the carioteca and the nuclear lamina, forming vesicles, or exaggerated chromatin condensation, resulting in pyknosis. In the cytoplasm vacuolization was also observed, besides organelle destruction.
The arrest of meiosis due to lock of chromosome homologies leads to germinative cell degeneration and, therefore, the spermatogenesis arrest. This fact causes a profound alteration in the seminiferous epithelium morphology in comparison with the parental species.     

Expression of nitric oxide synthase isoforms in the testes of pigs

This study examined the expression of three isoforms of nitric oxide synthase (NOS) in the testes of pigs. Immunohistochemical studies demonstrated the presence of nNOS, eNOS and iNOS in interstitial cells, primary spermatocytes and spermatids. Positive immunoreactions for eNOS and iNOS were detected in peritubular myoid cells. Some vascular endothelial cells were positive for nNOS and eNOS. The expression of nitrotyrosine was detected in interstitial cells. In addition, the histochemical study revealed that all the interstitial cells were stained positively for NADPH-diaphorase, although some spermatids and vascular endothelial cells displayed moderate enzymatic activity. These findings suggest that three isoforms of NOS are expressed in the testis of pig and that they play important roles in the biology of interstitial cells that produce testosterone, as well as in spermatogenesis in the seminiferous tubules.     

Clinical Use of Testicular Fine Needle Aspiration Cytology in Oligozoospermic and Azoospermic Dogs

Clinical investigation of canine testicular function is complicated by the difficulty in the evaluation of seminiferous tubules. Until recently, testicular biopsy was the only diagnostic option for dogs with persistent oligo/azoospermia. In human andrology, testicular fine needle aspiration (TFNA) is currently considered a useful method in the evaluation of azoospermia and severe oligozoospermia, and has long replaced classical biopsy to evaluate spermatogenesis. In order to verify its diagnostic efficacy for the clinical approach to canine oligo- or azoospermia, TFNA was performed in seven adult (two oligozoospermic and five azoospermic) dogs. After sedation, a fine (21–23 gauge) butterfly needle connected to a 50-ml syringe was inserted into each testicle; strong suction was applied and the aspirated fluid squirted on a glass slide, smeared out, air-dried and stained with a modified May–Grunwald–Giemsa. Under light microscopy, Sertoli cells (all those found in each investigated field) and spermatogenic cells (n = 100) were counted on each smear in order to differentiate spermatogonia, primary spermatocytes, secondary spermatocytes, early spermatids, late spermatids and spermatozoa, and calculate their relative percentages. Cytological analysis showed the following testicular pictures: normal spermatogenesis (compatible with obstruction of the seminal ducts), hypospermatogenesis, maturative disturbances and Sertoli cell-only syndrome. Two dogs with an obstructive lesion were treated with corticosteroids; one of them recovered and sired two litters of puppies.     

羊驼睾丸细胞凋亡及凋亡相关蛋白的定位

本研究旨在观察羊驼睾丸的出生后发育和精子发生过程中的细胞凋亡及凋亡相关蛋白Bcl2和Caspase3 的定位.取材新生、12月龄和24月龄羊驼的睾丸,用TUNEL法检测睾丸发育和精子发生过程的细胞凋亡,用免疫组织化学技术检测凋亡相关蛋白Bcl2和Caspase3在羊驼出生后发育和精子发生过程中的定位.结果显示在新生羊驼睾丸未检测到TUNEL阳性细胞,Caspase3和Bcl2表达于间质细胞,提示在新生期凋亡蛋白参与间质细胞凋亡的调节,为曲精小管的发育提供空间;12月龄羊驼睾丸TUNEL阳性细胞定位于曲精小管中央部分,Caspase3 和Bcl2定位于间质细胞和曲精小管中央生殖细胞,提示在青春期(12月龄)羊驼睾丸,细胞凋亡和凋亡相关蛋白参与曲精小管管腔形成的调节;24月龄羊驼睾丸TUNEL阳性细胞定位于精原细胞、精母细胞和精子细胞,Caspase3 和Bcl2定位于间质细胞和各个发育阶段的生精细胞,Caspase3阳性细胞在精原细胞最高,向精母细胞和精子细胞逐渐减少,Bcl2在精原细胞弱阳性表达,在血睾屏障以内的曲精小管近腔室部分呈弥散性强阳性表达,提示在性成熟(24月龄)羊驼睾丸精子发生过程中,细胞凋亡主要发生于精原细胞和早期精母细胞,Bcl2可能抑制精母细胞之后生殖细胞的凋亡.结果提示在羊驼睾丸出生后发育和精子发生过程中存在细胞凋亡现象;凋亡蛋白Caspase3和Bcl2参与羊驼睾丸发育和精子发生过程中细胞凋亡的调节.     

增精宝对环磷酰胺致小鼠生精障碍保护作用的研究

为评价增精宝对雄性动物生精作用的影响,采用环磷酰胺致小鼠生精障碍为动物模型,考察不同剂量增精宝对环磷酰胺致小鼠生精障碍的保护作用。结果显示,高剂量组对小鼠的附睾、睾丸、储精囊和前列腺均有较显著的促进作用;低剂量组精子总密度和精子活率均差异显著(P<0.05);高剂量组精子活率差异显著(P<0.05);对小鼠睾丸各倍体生精细胞比例的影响,高剂量组小鼠睾丸生精小管较为饱满,管腔内存在大量精子,生精上皮较厚,可见各级生精细胞排列整齐,界膜明显,睾丸间质细胞排列整齐。结论表明,增精宝两种剂量均能够显著促进环磷酰胺导致小鼠生精障碍的保护作用,高剂量组对雄性小鼠的作用尤为显著。