Stages Of Seminiferous Epithelium Research Articles

Gestational Exposure to Bisphenol A Affects Testicular Morphology, Germ Cell Associations, and Functions of Spermatogonial Stem Cells in Male Offspring.

Exposure to bisphenol A (BPA) in the gestational period damages the reproductive health of offspring; detailed evidence regarding BPA-induced damage in testicular germ cells of offspring is still limited. In this study, pregnant mice (F0) were gavaged with three BPA doses (50 μg, 5 mg, and 50 mg/kg body weight (bw)/day; tolerable daily intake (TDI), no-observed-adverse-effect-level (NOAEL), and lowest-observed-adverse-effect level (LOAEL), respectively) on embryonic days 7 to 14, followed by investigation of the transgenerational effects of such exposure in male offspring. We observed that the NOAEL- and LOAEL-exposed F1 offspring had abnormalities in anogenital distance, nipple retention, and pubertal onset (days), together with differences in seminiferous epithelial stages and testis morphology. These effects were eradicated in the next F2 and F3 generations. Moreover, there was an alteration in the ratio of germ cell population and the apoptosis rate in germ cells increased in F1 offspring at the LOAEL dose. However, the total number of spermatogonia remained unchanged. Finally, a reduction in the stemness properties of spermatogonial stem cells in F1 offspring was observed upon LOAEL exposure. Therefore, we provide evidence of BPA-induced disruption of physiology and functions in male germ cells during the gestational period. This may lead to several reproductive health issues and infertility in offspring.

Spermiogenesis, stages of seminiferous epithelium and variations in seminiferous tubules during active states of spermatogenesis in Yangzhou goose ganders

The past three decades revolutionized the goose industry in the world. China holds the world's largest goose breeds stock by 95% of the global total. To optimize the goose industry and cope with ever increasing poultry meat and egg demands, there is a dire need to focus on reproduction, as most geese breeds exhibit poor reproductive performance. The present study was conducted with the aim to add a contribution in the goose industry and research by the histological visualizing step wise development of germ cells during spermatogenesis by microscopy and a histological technique. Yangzhou goose is a synthetic breed developed by using local goose germplasm resources of China. It is popular in the Chinese goose industry due to high productivity and adaptability. This research evaluated the steps of spermiogenesis and stages along with morphological changes in the seminiferous epithelium in Yangzhou goose ganders. For the assessment of various stages of the seminiferous epithelium cycle, testis sections were embedded in molten paraffin wax. The initial steps of spermiogenesis were depicted by changes in acrosomic granules, whereas further stages were identified by nuclear morphological changes. Ten steps of spermiogenesis and nine stages of seminiferous epithelium were identified. Four types of spermatogonia Ad, Ap1, Ap2 and B were recognized. The results depicted a clear variation in the diameter of seminiferous tubules (ST), epithelium height (EH), luminal tubular diameter (LD), number of seminiferous tubules per field and the Johnsen score. Microscopy indicated that the stages of seminiferous epithelium were similar to other birds and mammals and the ST diameter, EH, LD and Johnsen score are positively correlated while the number of seminiferous tubules per field is negatively correlated with the ST diameter, EH, LD and Johnsen score. Fertility in Yangzhou ganders can further be improved by visualizing the histological development of germs cells in testis tissues during spermatogenesis after onset of breeding season and maturity. Our results suggest that Yangzhou ganders reach complete sexual maturity at 227 days of age.

Spermiogenesis, Stages of Seminiferous Epithelium and Variations in Seminiferous Tubules during Active States of Spermatogenesis in Yangzhou Goose Ganders.

Simple SummaryOptimum hatchability and fertility in the goose industry is inevitable without emphasizing on reproductive health of ganders. Determining stages of spermatogenesis is a critical factor in evaluating the age of maturity and reproductive health of animals. The current study was designed to evaluate the steps of spermiogenesis and stages along with morphological changes in seminiferous epithelium in Yangzhou goose ganders. The results of the study revealed that inside seminiferous epithelium, initial steps of spermiogenesis are depicted by changes in acrosomic granules, whereas further stages are identified by nuclear morphological changes. Furthermore, ten steps of spermiogenesis, nine stages of seminiferous epithelium and four types of spermatogonia Ad, Ap1, Ap2 and B were identified. In Yangzhou goose ganders, recommended age of sexual maturity is 227 days of age.The past three decades revolutionized the goose industry in the world. China holds the world’s largest goose breeds stock by 95% of the global total. To optimize the goose industry and cope with ever increasing poultry meat and egg demands, there is a dire need to focus on reproduction, as most geese breeds exhibit poor reproductive performance. The present study was conducted with the aim to add a contribution in the goose industry and research by the histological visualizing step wise development of germ cells during spermatogenesis by microscopy and a histological technique. Yangzhou goose is a synthetic breed developed by using local goose germplasm resources of China. It is popular in the Chinese goose industry due to high productivity and adaptability. This research evaluated the steps of spermiogenesis and stages along with morphological changes in the seminiferous epithelium in Yangzhou goose ganders. For the assessment of various stages of the seminiferous epithelium cycle, testis sections were embedded in molten paraffin wax. The initial steps of spermiogenesis were depicted by changes in acrosomic granules, whereas further stages were identified by nuclear morphological changes. Ten steps of spermiogenesis and nine stages of seminiferous epithelium were identified. Four types of spermatogonia Ad, Ap1, Ap2 and B were recognized. The results depicted a clear variation in the diameter of seminiferous tubules (ST), epithelium height (EH), luminal tubular diameter (LD), number of seminiferous tubules per field and the Johnsen score. Microscopy indicated that the stages of seminiferous epithelium were similar to other birds and mammals and the ST diameter, EH, LD and Johnsen score are positively correlated while the number of seminiferous tubules per field is negatively correlated with the ST diameter, EH, LD and Johnsen score. Fertility in Yangzhou ganders can further be improved by visualizing the histological development of germs cells in testis tissues during spermatogenesis after onset of breeding season and maturity. Our results suggest that Yangzhou ganders reach complete sexual maturity at 227 days of age.

Characterization of Seminiferous Epithelium Stages in the Wild Javan Muntjac (Muntiacus muntjak muntjak) Using the Tubular Morphology Method.

Stages of the seminiferous epithelium of the testis of the wild Javan muntjac (Muntiacus muntjak muntjak) in hard antler period were characterized based on the tubular morphology method. The number and the relative frequencies of seminiferous epithelium stages and the morphometry of germinal cell nuclei were identified microscopically. We identified eight stages of seminiferous epithelium in testicular tissue of the Javan muntjac and found that the relative frequencies of stages I to VIII were 14.87, 15.12, 17.75, 6.87, 7.37, 12.37, 13, and 12.62%, respectively. The diameter of the nuclei of germinal cells varied in each stage of seminiferous epithelium. Diplotene-stage primary spermatocytes had prominent and large nuclei ~8.97 ± 1.0 μm in stages III and IV. Pachytene primary spermatocytes appeared in most stages, except stage IV, whereas leptotene- and diplotene-stage primary spermatocytes were found in stages I and II, and III and IV, respectively. Round spermatids were observed in stages IV to VIII and in stage I but were absent in stages II and III, while elongated spermatids were observed in all stages except stage I. Our findings show that the stages of seminiferous epithelium in the Javan muntjac are similar to those found in neotropical cervids, small ruminants, and other domestic animals.

Stage-Specific Expression of CYP26B1 in the Adult Testis is Responsible for Pulsatile Retinoic Acid Signaling in Spermatogenesis

Retinoic acid (RA) plays important roles in spermatogenesis process; however, mechanisms underlying pulsatile RA signaling in meiotic initiation has not been understood yet. We studied expression pattern of RA-degrading enzyme CYP26B1 during development of rhesus monkey testis using qPCR and IHC. In developing monkey testis, the CYP26B1 protein was detected in cytoplasm of undifferentiated spermatogonia. Expression level of CYP26B1 mRNA was down-regulated during testis development which is consistent with initiation of meiosis in adult testis. In adult testes, a heterogeneous pattern of CYP26B1 expression was observed along different stages of seminiferous epithelium, presumably indicates expression pattern of protein is stage specific. Highest level of CYP26B1 protein expression was coincided with onset of meiosis and observed in early meiotic spermatocytes within seminiferous epithelial stages X-XII. Whereas, lowest level of CYP26B1 expression was observed in stages VI-IX of seminiferous epithelium, where undifferentiated Type A spermatogonia divide and differentiate to Type B spermatogonia, meiosis initiates and spermiogenesis occurs. This observations led us to suggest CYP26B1 might be responsible for pulsatile RA signaling in spermatogenesis. Findings presumably support that elevated amount of RA in undifferentiated Type A spermatogonia during stages VI-IX of the seminiferous epithelium of adult monkey testis may be responsible for differentiation of spermatogonia and their meiotic entry

Functional role of progestin and the progesterone receptor in the suppression of spermatogenesis in rodents

Synthetic progestins such as levonorgestrel (LNG) are used in combination with testosterone (T) in male contraceptive clinical trials to suppress gonadotropins secretion, but whether progestins have additional direct effects on the testis are not known. This study aimed to examine the effect of a potent progestin, (LNG), alone or in combination with testosterone (T) on spermatogenesis in adult rats, and to evaluate the functional role of the progesterone receptors (PRs) in the testis. In comparison with a low dose of LNG treatment in adult rats for 4 weeks, T and T + LNG treatment decreased testicular sperm count to 64.1 and 40.2% of control levels respectively. LNG induced germ cell apoptosis at stages I-IV and XII-XIV; T increased apoptosis at stages VII-VIII; LNG + T treatment induced greater germ cell apoptosis at a wider range of seminiferous epithelial stages. RT-PCR and Western Blots showed that PR was present in testes and up-regulated during suppression of spermatogenesis induced by testicular hormonal deprivation. PR knockout (PRKO) mice had larger testes, greater sperm production, increased numbers of Sertoli and Leydig cells. Suppression of gonadotropin and intratesticular T by GnRH-antagonist treatment induced PR promoter driven LacZ expression in Leydig cells of PRKO mice. This suggests that GnRH-antagonist treatment while inducing germ cell apoptosis also up-regulates PR. We conclude that (i) LNG + T induced greater suppression of spermatogenesis through increase in germ cell apoptosis involving a wider range of seminiferous epithelial stages than either treatment alone, (ii) up-regulation of PR was associated with inhibition of spermatogenesis, (iii) PR knockout mice showed increased sperm production suggesting that testicular PR activated events play a physiological and pharmacological inhibitory role in the testis. These data support the hypothesis that in addition to its known suppressive effects on gonadotropins, progestins may have direct inhibitory actions on the testis.

Jmjd1a Demethylase-regulated Histone Modification Is Essential for cAMP-response Element Modulator-regulated Gene Expression and Spermatogenesis

Spermatogenesis, a fundamental process in the male reproductive system, requires a series of tightly controlled epigenetic and genetic events in germ cells ranging from spermatogonia to spermatozoa. Jmjd1a is a key epigenetic regulator expressed in the testis. It specifically demethylates mono- and di-methylated histone H3 lysine 9 (H3K9me1 and H3K9me2) but not tri-methylated H3K9 (H3K9me3). In this study, we generated a Jmjd1a antibody for immunohistochemistry and found Jmjd1a was specifically produced in pachytene and secondary spermatocytes. Disruption of the Jmjd1a gene in mice significantly increased H3K9me1 and H3K9me2 levels in pachytene spermatocytes and early elongating spermatids without affecting H3K9me3 levels. Concurrently, the levels of histone acetylation were decreased in Jmjd1a knock-out germ cells. This suggests Jmjd1a promotes transcriptional activation by lowering histone methylation and increasing histone acetylation. Interestingly, the altered histone modifications in Jmjd1a-deficient germ cells caused diminished cAMP-response element modulator (Crem) recruitment to chromatin and decreased expression of the Crem coactivator Act and their target genes Tnp1 (transition protein 1), Tnp2, Prm1 (protamine 1), and Prm2, all of which are essential for chromatin condensation in spermatids. In agreement with these findings, Jmjd1a deficiency caused extensive germ cell apoptosis and blocked spermatid elongation, resulting in severe oligozoospermia, small testes, and infertility in male mice. These results indicate that the Jmjd1a-controlled epigenetic histone modifications are crucial for Crem-regulated gene expression and spermatogenesis.

Transcriptional Profiling of the Hormone-Responsive Stages of Spermatogenesis Reveals Cell-, Stage-, and Hormone-Specific Events

Spermatogenesis occurs within the highly complex seminiferous epithelium. This cyclic process is accompanied by dynamic stage-specific transcriptional changes and is driven by androgens and FSH by mechanisms that are unclear. Here we report the impact of acute androgen and FSH suppression on the transcriptional dynamics of the seminiferous epithelium. We used transcriptional profiling to compare the most hormone-sensitive seminiferous epithelial stages (VII and VIII) from control and hormone-suppressed adult rats, together with publicly available datasets to delineate stage- and cell-specific transcriptional changes. The analyses reveal that, in these stages, there was a hormone-responsive down-regulation of spermatogonial and Sertoli cell transcripts maximally expressed in the earlier spermatogenic stages (I-VI). Transcripts expressed in Sertoli cells from stage VII and beyond were both up- and down-regulated by hormone suppression, with lysosome function, immune system-related genes, and lipid metabolism predicted to be hormone responsive. Hormone-responsive genes with putative roles in integrin-mediated cell adhesion were also identified. In pachytene spermatocytes, there was an initiation of transcription likely important for the completion of meiosis. A transcriptional switch in round spermatids was observed, from a hormone-responsive down-regulation of transcripts expressed in steps 1-7 spermatids to a hormone-independent up-regulation of transcripts expressed in steps 8-11 and likely involved in spermatid differentiation and DNA compaction. This study points to the existence of hormone-responsive global transcriptional repressors in Sertoli cells, spermatogonia, and spermatids and reveals novel and diverse cell-specific responses of the seminiferous epithelium to hormone suppression.

Classification of the Seminiferous Epithelial Cycle in the Sika Deer (Cervus nippon)

The sika deer (Cervus nippon) is the only Cervidae inhabiting Japan, except a feral foreign cervid, the Reeves’ muntjac (Muntiacus reevesi). The sika deer is short-day seasonal breeder, and its rut occurs from October to December (Suzuki et al. 1992). When considering the biology of seasonal breeders, it is especially important to elucidate testicular functions during seasonal testicular changes. In the mature testes of the roe deer during the breeding season, the composition of the seminiferous epithelium periodically changes throughout spermatogenesis (Blottner et al. 1996). Many studies on the stages of the epithelial cycle have been reported in experimental and domestic animals (Foote et al. 1972; Swierstra et al. 1974; Berndtson 1977; Ekstedt et al. 1986; Pawar and Wrobel 1991). For example, the seminiferous epithelial cycle is divided into 14 stages in the rat (Leblond and Clermont 1952) and 8 stages in the bull (Wrobel and Schimmel 1989). Information about these stages is essential for morphological and functional researches on the testes and provides the basis of understanding testicular regulatory mechanisms (Toppari et al. 1986; Zhu et al. 1997; Zhou et al. 2002; Schon et al. 2004; Klonisch et al. 2006). In the rat, the morphological changes of the Sertoli cells with each seminiferous epithelial stage are concerned with the control of phosphorylation of the cytoskeletal protein, vimentin (Zhu et al. 1997). In the mouse, androgen receptors in the Sertoli cells show stage-dependent expression with the most intense expression in stages VI–VII (Zhou et al. 2002). Histological studies on postnatal testicular development and seasonal changes of the testes have been reported in the sika deer (Yamauchi et al. 1982). Structural changes of the seminiferous tubules, plasma and fecal testosterone levels and localization of steroidogenic enzymes have already been examined with annual testicular changes of the sika deer (Suzuki et al. 1992; Yamauchi et al. 1997; Kameyama et al. 2002; Hayakawa et al. 2004). However, the stages of the seminiferous epithelial cycle are not yet fully defined in the sika deer, although they have been reported in other cervids, the fallow deer (Wrobel et al. 1993) and roe deer (Schon et al. 2004). In the present study, therefore, we categorized the seminiferous epithelial stages in the sika deer.

Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) in Spermatogenic Cells Induced by Retinoic Acid: An In Vivo Study in Vitamin A-Sufficient Postnatal Murine Testes1

Vitamin A is required for male fertility and normal spermatogenesis. Retinoic acid (RA), an active metabolite of vitamin A, is necessary for spermatogonial maturation and proper entry of germ cells into meiotic prophase in the postnatal testes. The expression of Stra8, which is essential for successful meiosis in both male and female gonads and normal spermatogenesis, is directly related to the availability of RA. This study examined the developmental expression pattern of Stra8 transcript in both male and female gonads, provided specific cellular localization of STRA8 protein in the postnatal and adult testis, and investigated RA actions in adult germ cells in a vitamin A-sufficient condition. The peak of Stra8 mRNA expression coincided with the onset of meiosis in postnatal testes. STRA8 protein was detected in gonocytes as early as 5 days postpartum. The expression of STRA8 protein in the neonatal testes was not uniform among spermatogonia, perhaps heralding the asynchronous beginning of spermatogenesis. In adult testes, the highest level of Stra8 mRNA and protein was found in seminiferous epithelial stages VI-VIII. STRA8 protein was localized to some type A and B spermatogonia, preleptotene spermatocytes, and early leptotene spermatocytes. In the vitamin A-sufficient adult testes, RA but not retinol acetate stimulated Stra8 mRNA expression. STRA8 protein expression in adult spermatogonia was induced by RA stimulation, suggesting its role in spermatogonial differentiation. Retinoic acid also increased the number of preleptotene spermatocytes exhibiting 5-bromo-2-deoxyuridine incorporation, indicating a more synchronized premeiotic DNA replication.

Histological description of seminiferous epithelium and cycle length of spermatogenesis in the water shrew Neomys fodiens (Mammalia: Soricidae)

Recently, we examined the spermatogenesis cycle length in two shrews species, Sorex araneus characterized by a very high metabolic rate and a polyandric mating system (sperm competition) resulting in a short cycle and Crocidura russula characterized by a much lower metabolic rate and a monogamous mating system showing a longer cycle. In this study, we investigated the spermatogenesis cycle in Neomys fodiens showing an intermediate metabolic rate. We described the stages of seminiferous epithelium according to the spermatid morphology method and we calculated the cycle length of spermatogenesis using incorporation of 5-bromodeoxyuridine into DNA of the germ cells. Twelve males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determination, we applied a recently developed statistical method. The calculated cycle length is 8.69 days and the total duration of spermatogenesis based on 4.5 cycles is approximately 39.1 days, intermediate between the duration of spermatogenesis of S. araneus (37.6 days) and C. russula (54.5 days) and therefore congruent with both the metabolic rate hypothesis and the sperm competition hypothesis. Relative testes size of 1.4% of body mass indicates a promiscuous mating system.

Reduction of fertility in the male rat by systemic treatment with follicle regulatory protein

Forty-five-day-old rats received daily injections of follicle regulatory protein (FRP). After 15, 30, 45, and 70 days of therapy, serum was measured for testosterone, androstenedione, estradiol, and follicle-stimulating hormone levels. Testes were evaluated for sperm head counts, plasminogen activator activity, weight, and length of seminiferous epithelial stages. In no case was serum follicle-stimulating hormone concentration reduced in FRP-treated rats. After 75 days of treatment, there was a significant decrease in the number of the sperm head counts. After 60 days of treatment, the length of the dark zone of the tubule was longer than that of control. Pregnancy rates for FRP-treated rats were reduced after 45 and 60 days of treatment. In conclusion, systemic injection of FRP alters seminiferous epithelial function by reducing development of mature sperm.

Disruption of seminiferous epithelial function in the rat by ovarian protein.

The granulosa cell produces an inhibitor of aromatase activity, which recently was purified to homogeneity (follicle-regulatory protein: FRP). Since extracts of testicular homogenates also contain factor(s) with biological properties similar to FRP, including inhibition of granulosa cell aromatase, we examined the effects of ovarian FRP on testicular function. Forty-five-day-old rats received daily FRP injections (100 micrograms or 300 micrograms). After 15, 30, 45, and 70 days of therapy, (n = 5 each group), trunk serum was measured for testosterone, androstenedione, estradiol, and FSH levels by established radioimmunoassays (RIA). One testis from each rat was homogenized, centrifuged, and evaluated for sperm head counts; the other testis was dissected by transillumination, and the length of seminiferous epithelial stages determined. After 15 (control: 4.8 +/- 0.2 mm; 100 micrograms: 6.0 +/- 0.3 mm; 300 micrograms: 6.6 +/- 0.3 mm) and 30 days (control: 4.6 +/- 0.2 mm; 100 micrograms: 6.3 +/- 0.2 mm; 300 micrograms: 5.9 +/- 0.2 mm) of treatment the length of the "strong" seminiferous tubule segment in FRP-treated rats was greater than in control rats (p less than 0.05). Serum levels of steroids and FSH were similar in all groups. After 30, 45, and 70 days of treatment, the sperm head counts for the 100-micrograms and 300-micrograms dosages were 26%, 29%, 30% and 20%, 34%, and 24% of control values, respectively. By 70 days of treatment, cycle Stage VII was markedly reduced or absent in FRP-treated rats, and their round spermatids contained ring chromatin; both conditions indicate degeneration. FRP (50 micrograms/ml) was added to rat Sertoli cell cultures for 4 days after which transferrin and androgen-binding protein (ABP) were measured. FRP enhanced Sertoli cell secretion of ABP (58 vs. 138 +/- 7 microliters eq/culture) and transferrin (2.1 vs. 4.7 +/- 0.6 microgram/culture). In conclusion, systemic injection of FRP alters seminiferous epithelial function by reducing maturation of mature sperm forms. Adding FRP to Sertoli cells in culture enhances secretion of transferrin and ABP; this suggests that maturation of the germinal elements may be linked to the secretory function of seminiferous tubules.