Assessment of spermatogenesis in camels via seminiferous tubule staging and testosterone profiling

Objective:To investigate the successive morphological stages of spermatogenesis,hormonal regulation,and testosterone profile in dromedary camel reproduction.Methods:Testicular tissue samples were obtained from 12 dromedary bull camels aged 7 to 8 at a local abattoir.The histological assessment involved tissue processing,hematoxylin and eosin(H&E)staining,and examination under a microscope.Stereological analysis,germ cell identification,and assessment of seminiferous tubules and maturation were conducted.Testosterone assay was performed by radioimmunoassay using blood samples collected at regular intervals.Results:The study revealed 12 phases of the dromedary camel's seminiferous epithelium cycle,highlighting distinct morphological characteristics and cellular processes.Acrosomal migration,maturation,cap formation,and the Golgi-mediated synthesis of proacrosomal vesicles were also explained in dimension,as were the steps of acrosome biogenesis.Spermatids and mature sperm cells were present when spermatogenesis phases were examined.An analysis of the dimensions of seminiferous tubules revealed specific measures for diameter,area,and epithelial height about luminal characteristics.Moreover,there were noticeable variations in the serum testosterone concentrations during the study period,indicating temporal dynamics.Conclusions:This study outlines the spermatogenesis process in dromedary camels across 12 stages,emphasizing cellular dynamics and acrosomal biogenesis.It also provides seminiferous tubule measurements and observes seasonal testosterone fluctuations,offering insights into reproductive regulation and potential strategies for camel breeding conservation.

Testis Development and Ultrastructural Features During Spermatogenesis in Cultured Small Yellow Croaker,Larimichthys polyactis

The small yellow croaker Larimichthys polyactis is an economically important marine fish in Northeast Asia.Currently,its natural resources are threatened by overfishing and environmental pollution.Therefore,research on the reproductive system of the fish is crucial.Here,we studied the testis development and ultrastructural features of spermatogenesis in cultured L.polyactis using anatomical,histological,and ultrastructural techniques.A pair of testes,consisting of a central sperm duct and radial seminiferous lobules,were observed.The reproduction cycle of testes can be divided into stages I–VI.March to May was confirmed as the breeding season for male L.polyactis,while April is the ideal period for artificial breeding.The male L.polyactis can attain sexual maturity within 1 year.The spermatogenesis of L.polyactis comprised spermatogonium,spermatocyte,spermatid,and mature spermatozoon.The morphology of spermatogenic cells changed obviously during spermiogenesis,including nuclear shaping,midpiece and flagellum formation.The mature sperms consist of an ellipsoidal head,a short midpiece,and a long flagellum.The anterior of the head with a kidney-shaped nucleus can be distinguished.The midpiece is located posterior to the head and includes four to six spherical mitochondria.The flagellum has irregular lateral fins.The testis of L.polyactis is an unrestricted lobular type,with cystic spermatogenesis,type II spermiogenesis,and type II spermatozoa.These features are highly similar to those of other Sciaenid species.Our findings provide useful insights into the mechanism underlying testis development and spermatogenesis of L.polyactis,which can facilitate the artificial breeding of this species.

The effects of hormone-mediated PI3K/AKT signaling on spermatogenesis in Sertoli cells

The phosphoinositide-3-kinase/Akt(PI3K/AKT)signaling pathway is crucial for Sertoli cell development and completing spermatogenesis.Its main role is to promote proliferation and inhibit apoptosis.Many factors activate the PI3K/AKT pathway,like hormones,such as follicle stimulating hormone(FSH),androgen,estrogen,insulin to name a few.Many of these factors have receptors inside or on the surface of Sertoli cells(SCs).This review summarizes how these hormones directly regulate the PI3K/AKT signaling pathway in SCs,which in turn affects SC proliferation and differentiation.Further,hormone-mediated PI3K/AKT signaling also stimulates SC secretion,which is essential for germ cell development,suggesting an indirect role of PI3K/AKT signaling during spermatogenesis.These functions include promoting spermatogonia proliferation and differentiation,meiosis of spermatocytes,sperm maturation,and their release.This review also provides potential hints for clinically treating male infertility issues like cryptorchidism and Sertoli cell-only syndrome.

Effect of sodium arsenite on spermatogenesis, plasma gonadotrophins and testosterone in rats

<abstract> To investigate the effect of arsenic on spermatogenesis. Methods: Mature (4 months old) Wistar rats were intraperitoneally administered sodium arsenite at doses of 4, 5 or 6 mg-kg^-day1 for 26 days. Different varieties of germ cells at stage VII seminiferous epithelium cycle, namely, type A spermatogonia (ASg), preleptotene spermatocytes (pLSc), midpachytene spermatocytes (mPSc) and step 7 spermatids (7Sd) were quantitatively evaluated, along with radioimmunoassay of plasma follicle-stimulating hormone (FSH), lutuneizing hormone (LH), testosterone and assessment of the epididymal sperm count. Results: In the 5 and 6 mg/kg groups, there were significant dose-dependent decreases in the accessory sex organ weights, epididymal sperm count and plasma concentrations of LH, FSH and testosterone with massive degeneration of all the germ cells at stage VII. The changes were insignificant in the 4 mg/kg group. Conclusion: Arsenite has a suppressive influence on spermatogenesis and gonadotrophin and testosterone release in rats.

Regulation of spermatogenesis by paracrine/autocrine testicular factors

Spermatogenesis is a complex process regulated by endocrine and testicular paracrine/autocrine factors. Gonadotropins are involved in the regulation of several testicular paracrine factors, mainly of the IL-1 family and testicular hormones. Testicular cytokines and growth factors (such as IL-1, IL-6, TNF, IFN-γ, LIF and SCF) were shown to affect both the germ cell proliferation and the Leydig and Sertoli cells functions and secretion. Cytokines and growth factors are produced by immune cells and in the interstitial and seminiferous tubular compartments by various testicular cells, including Sertoli, Leydig, peritubular cells, spermatogonia, differentiated spermatogonia and even spermatozoa. Corresponding cytokine and growth factor receptors were demonstrated on some of the testicular cells. These cytokines also control the secretion of the gonadotropins and testosterone in the testis. Under pathological conditions the levels of pro-inflammatory cytokines are increased and negatively affected spermatogenesis. Thus, the expression levels and the mechanisms involved in the regulation of testicular paracrine/autocrine factors should be considered in future therapeutic strategies for male infertility.

Effect of Lepidium meyenii (maca) roots on spermatogenesis of male rats

Aim: To determine the effect of oral administration of an aqueous extract from the roots of Lepidium meyenii (maca)on spermatogenesis in adult male rats. Methods; Male rats received an aqueous extract of the root (66.7 mg in onemL) twice a day for 14 consecutive days. Results: Treatment with Lepidium meyenii resulted in an increase in theweights of testis and epididymis but not the seminal vesicle weight. The length and frequency of stages IX-XIV seminif-erous tubules, where mitosis occurred, were increased and stages I-VI were reduced in rats treated with Lepidiummeyenii. Conclusion; The Lepidium meyenii root invigorates spermatogenesis in male rats by acting on its initialstages (IX-XIV). (Asian J Androl 2001 Sep; 3: 231 - 233)

Expression and localization of CKLFSF2 in human spermatogenesis

Aim： To investigate the expression and subcellular localization of chemokine-like factor superfamily 2 （CKLFSF2） in human testis and its potential role in spermatogenesis. Methods： A specific polyclonal antibody against CKLFSF2 was raised. The expression and cellular localization of CKLFSF2 in the seminiferous tubules was checked by immunohistochemistry method. Also, in situ hybridization was applied to localize the mRNA distribution. The EGFP- CKLFSF2 fusion protein was expressed in COS-7 cells to localize its subcellular location in vitro. In addition, the abnormal expression of CKLFSF2 in testes of patients with male infertility was assayed by reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry methods. Results： Having a close correlation with spermatogenesis defects, CKLFSF2 was specifically expressed in meiotic and post-meiotic germ cells, which were localized to the endoplasmic reticulum （ER） near the Golgi apparatus. Conclusion： CKLFSF2 could play important roles in the process of meiosis and spermiogenesis, and might be involved in the vesicular transport or membrane apposition events in the endoplasmic reticulum.

Blood-testis barrier and spermatogenesis： lessons From genetically-modified mice

The blood-testis barrier （BTB） is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions （TJs）, adhesion junctions and gap junctions （GJs）. Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.

Effect of vasectomy via inguinal canal on spermatogenesis in rabbits

Aim： To determine whether vasectomy away from the epididymal tail （via the inguinal canal） in rabbits can reduce the early postoperative effects on spermatogenesis. Methods： Twenty-nine normal male Japanese white rabbits （aged 4- 6 months） were subjected to unilateral close-ended （conventional） or open-ended （the cut end of the juxta-epididymal vas deferens not ligated） vasectomy via the inguinal canal. Ten days and 3 months after operation, testes, epididymides and vasa deferentia were removed and methacrylate resin-embedded sections prepared. The histology of the testis, epididymis and vas deferens was examined under light microscope, and the volume and diameter of the seminiferous tubules were quantitatively studied using stereological methods. Results： Neither of the methods of vasectomy led to apparent damage to spermatogenesis on the vasectomized side in comparison with the contralateral shamoperated side, but the juxta-epididymal vas deferens on the vasectomized side was highly distended and contained numerous sperm 3 months after operation. Conclusion： Vasectomy away from the cauda epididymis has no significant early postoperative effects on spermatogenesis in rabbits.

Radio-protective effect of vitamin E on spermatogenesis in mice exposed to γ-irradiation: a flow cytometric study

Aim: To investigate the effect of vitamin E on the radioprotection of spermatogenesis and chromatin condensation of spermatozoa during passage through the epididymis in mice exposed to irradiation. Methods: Adult outbred male ICR mice were orally administered natural vitamin E (VE, D-α-tocopheryl acetate) at 400 IU/kg for 7 days before exposure to 1 Gy of γ-irradiation. The animals were sacrificed at day 1, 7, 14, 21, 28, 35 and 70 post-irradiation (IR) and the percentage of testicular germ cells and epididymal sperm chromatin condensation was analyzed using flow cytometry. Results: Serum D-α-tocopheryl acetate levels were 47.4 ± 3.2 μg/dL in the treated group, yet it could not be detected in the control group. The testicular weight of irradiated mice pretreated with VE+IR was significantly (P<0.05) higher than that of those without VE treatment (IR) at day 14 and 21 post-irradiation. The percentage of primary spermatocytes (4C) in the VE+IR group was comparable to the controls but significantly (P<0.05) higher than those in the IR group from day 7 to 35 post-irradiation. The percentage of round spermatids (1C) in the VE+IR group was also significantly (P<0.05) higher than those in the IR group at day 28 post-irradiation. The primary spermatocytes : spermatogonia ratio in the IR group was significantly (P<0.05) declined at day 7 to 35 post-irradiation when compared to the VE+IR and control groups. The round spermatid : spermatogonia ratio in the VE+IR group was significantly (P<0.05) higher than that of the IR group at day 14 and 28 post-irradiation. The chromatin condensation of epididymal spermatozoa measured by propidium iodide uptake was not affected by 1 Gy of γ-irradiation. Conclusion: The administration of VE prior to irradiation protects spermatogenic cells from radiation.

Preliminary study of letrozole use for improving spermatogenesis in non-obstructive azoospermia patients with normal serum FSH

We investigated whether letrozole （2.5 mg day-1） improves sperm count in non-obstructive azoospermia （NOA） patients. Four men were included in this study, and they had folliculo-stimulating hormone and other hormone levels within the normal range and no varicoceles or chromosomal aberrations. These four patients were administered letrozole for 3 months. Sperm count, testicular volume, gonadotropin, testosterone （T） and estradiol （E2） blood levels were assessed before, during and 1 week after the suspension of treatment. All patients showed spermatozoa in their ejaculate, increased gonadotropin and T levels and lower E2 levels （P〈0.05 in all cases）, when letrozole was administered. This suggests that letrozole treatment might improve sperm count in an NOA sub-population; however, more studies, including the proper controls, are needed to confirm its efficacy.

Proteomics of spermatogenesis： from protein lists to understanding the regulation of male fertility and infertility

Proteomic technologies have undergone significant development in recent years, which has led to extensive advances in protein research. Currently, proteomic approaches have been applied to many scientific areas, including basic research, various disease and malignant tumour diagnostics, biomarker discovery and other therapeutic applications. In addition, proteomics-driven research articles examining reproductive biology and medicine are becoming increasingly common. The key challenge for this field is to move from lists of identified proteins to obtaining biological information regarding protein function. The present article reviews the available scientific literature related to spermatogenesis. In addition, this study uses two-dimensional electrophoresis mass spectrometry （2DE-MS） and liquid chromatography （LC）-MS to construct a series of proteome profiles describing spermatogenesis. This large-scale identification of proteins provides a rich resource for elucidating the mechanisms underlying male fertility and infertility.

Reversible effect of testosterone undecanoate injection on spermatogenesis in rats

Aim: To study the effect of testosterone undecanoate (TU) injection on spermatogenesis in rats. Methods:Twenty adult SD rats received vehicle or TU (8 mg/kg, 19 mg/kg or 625 mg/kg) injection, im, every 15 days for 60days, and another 38 animals received similar treatments for 130 days with half of them undergoing a recovery phase of120 days (5 rats for each treatment). At the end of the treatment, testes were removed and the diameter of the seminif-erous tubules and the number of late elongated spermatids (steps 15-19) per testis were estimated with stereologicalmethods as a measure of the spermatogenic efficiency. Results: Low dose (8 mg/kg) TU treatment virtually hadno effect on spermatogenesis. A dose of 19 mg/kg slightly suppressed spermatogenesis 60 days after treatment, and se-vere suppression occurred after another 70 days of dosing. Spermatogenesis was completely recovered at the end of therecovery phase. Large dose (625 mg/kg) TU treatment did not significantly affect spermatogenesis and was well toler-ated by animals. Conclusion: TU injection reversibly suppresses spermatogenesis in rats.

The role of C-type natriuretic peptide in rat testes during spermatogenesis

C-type natriuretic peptide （CNP） is a 22-amino acid peptide and act as a local paracrine or autocrine regulator. There is growing evidence that ChIP is involved in male reproductive processes. To investigate the role of CNPduring spermatogenesis, we measured the mRNA expression of CNPand its specific membrane-bound natriuretic peptide receptor-B （NPR-B） using real-time RT-PCR in the testes of normal rats on different postnatal days. After that spermatogenesis dysfunction model induced by ornidazole was established with the aim to study the correlation of CNPwith spermatogenic dysfunction. Then, Sertoli cells from 18- to 22-day-old healthy male rats were cultured in the presence of different CNPconcentrations （1 ×10-6, 1×10-7 and1×10-8 mol l-1）, and the mRNA expression levels of androgen.binding protein, inhibin B and transferrin were examined at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. During the postnatal development of rat testes, the highest mRNA expression levels of CNPand NPR-B were found at postnatal Do, and the levels then declined gradually, with a second ChIPpeak at postnatal D35. In the ornidazole-induced infertile rat testes, CIVPgene expression was lower than in the uninduced rats （P〈0.05）, while IVPR-Bgene expression was greater （P〈0.05）. In cultured Sertoli cells, supplementation with CNP stimulated the gene expression of androgen-binding pmteirginhibin B/transferrin, particularly at 12 h, and 1× 10-7 mol l-1 CNP had the highest upregulation effect. The gene expression levels of CNPIIVPR-B in rat testes at different postnatal stages and in infertile rat testes indicated that CNP may participate in the physiology and/or pathology related to spermatogenesis. Moreover, ChIP regulated endocrine function in Sertoli cells. Taken together, these results showed that CNP is closely tied to spermatogenesis.

Evaluation of spermatogenesis and fertility in F1 male rats after in utero and neonatal exposure to extremely low frequency electromagnetic fields

Aim:To determine whether in utero and neonatal exposure to a 60 Hz extremely low frequency electromagnetic field (EMF) results in spermatotoxicity and reproductive dysfunction in the F1 offspring of rats.Methods:Age-matched, pregnant Sprague-Dawley rats were exposed continuously (21 h/day) to a 60 Hz EMF at field strengths of 0 (sham control),5,83.3 or 500 μT from day 6 of gestation through to day 21 of lactation.The experimentally generated magnetic field was monitored continuously (uninterrupted monitoring over the period of the study) throughout the study.Results:No exposure-related changes were found in exposed or sham-exposed animals with respect to the anogenital distance,preputial separation,testis weight,testicular histology,sperm count,daily sperm production, sperm motility,sperm morphology and reproductive capacity of F1 offspring.Conclusion:Exposure of Sprague- Dawley rats to a 60 Hz EMF at field strengths of up to 500 μT from day 6 of gestation to day 21 of lactation did not produce any detectable alterations in offspring spermatogenesis and fertility.

Analysis of the genetic interactions between Cyclin A1, Atm and p53 during spermatogenesis

Aim： To analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double- strand break repair. Methods： Two lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 （Ccnal）; p53- and Ccnal; Atm-double knockout mice. Results： The block in spermatogenesis observed in Cyclin A1-/- （Ccnal-/-） testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccnal-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccnal-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1. Conclusion： Ccnal-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.

Identification of a novel testis-specific gene and its potential roles in testis development/spermatogenesis

Aim:To identify and characterize a novel gene with potential roles in testis development and spermatogenesis. Methods:A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes.Differentially expressed genes were isolated and sequenced.RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis.A range of bioinformatical programs including Gene Runner,SMART,NCBI Blast and Emboss CpGPlot were used to characterize the new gene's feature.Results:A novel testis-specific gene, NYD-SP5,was differentially expressed in fetal and adult testes.The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved lie and Gin residues),a Carbam- ate kinase-like domain,a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain.RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined.In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes.Conclusion:NYD-SP5 is a newly found testis- specific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.

Identification and characterization of the BGR-like gene with a potential role in human testicular development/spermatogenesis

Aim: To investigate the roles of the BGR-like gene in testicular development/spermatogenesis. Methods: A human testis cDNA microarray was hybridized with probes from human adult testes and embryo testes. The differentially expressed clones were sequenced and analyzed. Expression of the BGR-like gene was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Results: A new gene exhibiting 50-fold difference in expression level between adult and fetal human testes was cloned and named the BGR-like gene. The cDNA consisted of 2500 nucleotides and had an open reading frame of 1437 nucleotides encoding a putative protein of 497 amino acid residues. Homologous comparison showed that the BGR-like gene was a new alternative splicing variant of the BGR gene and had sequence homology with the bubblegum gene of human, mouse, rat and Drosophilia. Protein motif analysis of the BGR-like gene revealed that it contained a conserved adenosine monophosphate (AMP)-binding domain and a fatty acyl-CoA synthetase signature motif which existed in all acyl-CoA synthetases. The BGR-like gene transcript was imperceptibly expressed in human fetal testes, highly in human adult testes and moderately in elderly testes and human Leydig cells. RT-PCR-based tissue distribution experiments showed that the BGR-like gene was exclusively expressed in testes and was a testes-specific isoform of the BGR gene. A BGR-like gene transcript was not detected in some azoospermic testes. Conclusion: The BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.

Low dose bisphenol A impairs spermatogenesis by suppressing reproductive hormone production and promoting germ cell apoptosis in adult rats

Bisphenol A （BPA）, an estrogenic chemical, has been shown to reduce sperm count; however, the underlying mechanisms remain unknown. Herein, we show that oral administration of BPA （2 μg/kg） for consecutive 14 days in adult rats （BPA rats） significantly reduced the sperm count and the number of germ cells compared to controls. The serum levels of testosterone and follicle-stimulating hormone （FSH）, as well as the level of GnRH mRNA in BPA rats were lower than those of control rats. Testosterone treatment could partially rescue the reduction of germ cells in BPA rats. Notably, the number of apoptotic germ cells was significantly increased in BPA rats, which was insensitive to testosterone. Furthermore, the levels of Fas, FasL and caspase-3 mRNA in the testicle of BPA rats were increased in comparison with controls. These results indicate that exposure to a low dose of BPA impairs spermatogenesis through decreasing reproductive hormones and activating the Fas/FasL signaling pathway.

Long-term effect of vasectomy on spermatogenesis in ,en： a morphometric study

Spermatogenic damage may occur after vasectomy, and the damage is pressure mediated, occurring when the occluded reproductive tract is unable to accommodate additional sperm produced by the testis. This study aimed to determine the long-term effect of vasectomy on spermatogenesis in humans and clarify how the balance between sperm production in the testis and sperm storage in or removal from the tract might be maintained. During inguinal hernia repair, an open biopsy was performed to obtain testicular tissue blocks from 51 Chinese men （aged ≥ 50 years）, of whom 25 （control group） had not undergone vasectomy and 26 （vasectomized group） had undergone bilateral vasectomy 22-42 years before. Methacrylate resin-embedded testicular sections were made, and morphometric studies were performed using light microscopy. In addition, sizes of the testis and epididymis were estimated with ultrasonography. The testicular tissue blocks obtained from one control and seven vasectomized men consisted almost completely of connective tissue. In the other 43 men, significant differences were not found between the two groups in the testicular or epididymal size, qualitative histology or quantitative parameters including the mean diameter or volume fraction of the seminiferous tubules. In conclusion, sperm production and sperm storage/removal reached a static equilibrium after vasectomy, likely due to spermatogenic degeneration or less sperm production as a result of aging or due to vasectomy-induced testicular （interstitial） fibrosis. Thus, complications that might occur in association with overproduction of sperm and distension of the tract would disappear or be relieved with time.