Targeting lentiviral vectors to primordial germ cells(PGCs):An efficient strategy for generating transgenic chickens

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

Proliferation of exogenously injected primordial germ cells (PGCs) into busulfan-treated chicken embryos

Aim: This study was designed to investigate the effect of busulfan treatment on the proliferation of chicken primordialgerm cells (PGCs) in vivo, focusing on the preferential settlement of PGCs onto the germinal ridges of chicken em-bryos. Methods: Busulfan (250 ng/egg) was injected into the egg white of freshly oviposited fertilized eggs, whichwere then incubated. Embryonic development and viability were examined, and exogenous PGCs collected from embry-onic blood vessels were injected into the germinal crescent region of recipient embryos. The number of PGCs residedonto germinal ridges of the right and left sides were compared. Results: Busulfan had a slight harmful effect on theembryo viability and the PGCs proliferation. The number of PGCs resided onto the left side of germinal ridges wasslightly higher as compared with the right side. Conclusion: Busulfan suppressed the viability of embryos and the pro-liferation of endogenous PGCs in the recipient embryos. However, the number of exogenous PGCs proliferated washigher in embryos treated with busulfan than those without busulfan. Data also suggest the possibility of a preferentialresidence of PGCs toward the left side of the germinal crescent region as compared with the fight, which may be due toa more advanced functional development of the left gonad than the right. (Asian J Androl 1999 Dec; 1: 187-190)

Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells

Background:NANOG is a core transcription factor(TF)in embryonic stem cells(ESCs)and primordial germ cells(PGCs).Regulation of the NANOG gene by TFs,epigenetic factors,and autoregulatory factors is well characterized in ESCs,and transcriptional regulation of NANOG is well established in these cells.Although NANOG plays a key role in germ cells,the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied.Therefore,we investigated the mechanism that regulates transcription of the chicken NANOG(cNANOG)gene in PGCs and ESCs.Results:We first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis.Then,we measured the promoter activity of various 5′flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay.cNANOG expression required transcriptional regulatory elements,which were positively regulated by POU5F3(OCT4)and SOX2 and negatively regulated by TP53 in PGCs.The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element(CCAAT/enhancer-binding protein(CEBP)-binding site)in ESCs.Furthermore,small interfering RNA-mediated knockdown demonstrated that POU5F3,SOX2,and CEBP played a role in cell type-specific transcription of cNANOG.Conclusions:We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner.This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

α-ketoglutarate promotes the specialization of primordial germ cell-like cells through regulating epigenetic reprogramming

There is growing evidence that cellular metabolism can directly participate in epigenetic dynamics and consequently modulate gene expression.However,the role of metabolites in activating the key gene regulatory network for specialization of germ cell lineage remains largely unknown.Here,we identified some cellular metabolites with significant changes by untargeted metabolomics between mouse epiblast-like cells(EpiLCs)and primordial germ cell-like cells(PGCLCs).More importantly,we found that inhibition of glutaminolysis by bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide(BPTES)impeded PGCLC specialization,but the impediment could be rescued by addition ofα-ketoglutarate(αKG),the intermediate metabolite of oxidative phosphorylation and glutaminolysis.Moreover,adding aKG alone to the PGCLC medium accelerated the PGCLC specialization through promoting H3 K27 me3 demethylation.Thus,our study reveals the importance of metabolite aKG in the germ cell fate determination and highlights the essential role of cellular metabolism in shaping the cell identities through epigenetic events.

Regulatory elements and transcriptional control of chicken vasa homologue(CVH)promoter in chicken primordial germ cells

Background： Primordial germ cells（PGCs）, the precursors of functional gametes, have distinct characteristics and exhibit several unique molecular mechanisms to maintain pluripotency and germness in comparison to somatic cells. They express germ cel-specific RNA binding proteins（RBPs） by modulating tissue-specific cis-and trans-regulatory elements. Studies on gene structures of chicken vasa homologue（CVH）, a chicken RNA binding protein, involved in temporal and spatial regulation are thus important not only for understanding the molecular mechanisms that regulate germ cel fate, but also for practical applications of primordial germ cells. However, very limited studies are available on regulatory elements that control germ cel-specific expression in chicken. Therefore, we investigated the intricate regulatory mechanism（s） that governs transcriptional control of CVH.Results： We constructed green fluorescence protein（GFP） or luciferase reporter vectors containing the various 5′ flanking regions of CVH gene. From the 5′ deletion and fragmented assays in chicken PGCs, we have identified a CVH promoter that locates at-316 to ＋275 base pair fragment with the highest luciferase activity. Additional y, we confirmed for the first time that the 5′ untranslated region（UTR） containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs. Furthermore, using a transcription factor binding prediction, transcriptome analysis and siR NA-mediated knockdown,we have identified that a set of transcription factors play a role in the PGC-specific CVH gene expression.Conclusions： These results demonstrate that cis-elements and transcription factors localizing in the 5′ flanking region including the 5′ UTR and an intron are important for transcriptional regulation of the CVH gene in chicken PGCs. Final y,this information wil contribute to research studies in areas of reproductive biology, constructing of germ cel-specific synthetic promoter for tracing primordial germ cells as wel as understanding the transcriptional regulation for maintaining germness in PGCs.

Chicken Mesenchymal Stem Cells as Feeder Cells Facilitate the Cultivation of Primordial Germ Cells from Circulating Blood and Gonadal Ridge

Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.

Developmental potency of mouse primitive ectoderm cells, embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.) mouse embryos and primordial germ cells from 12.50-day p.c.male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c.blastocysts.Sixteen (61.5) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells;four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts.However,no overt chimaera was obtained with either 5.25-day or 6.25-day embryonic ectoderm cells or 12.50-day male primordial germ cells.GPI analysis of mid-gestation conceptuses developed from injected blastocysts showedthat 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus.Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells,and their developmental potency becomes limited within a short period of time in normal development.

Effects of 17beta-estradiol on cell migration in male chicks distribution of primordial germ

Aim： To assess whether exogenous estradiol has any effect on migration of primordial germ cells （PGCs） in the chick. Methods： Fertilized eggs were treated with 17beta-estradiol （E2） （80 lag/egg） at stage X （day 0 of incubation）, stages 8-10 （incubation 30 h） and 13-15 （incubation 55 h）. Controls received vehicle （emulsion） only. Changes in PGC number were measured on different days according to developmental stages. Results： In male right gonads, but not in female left gonads, at stages 28-30 （incubation 132 h） significant decreases in the mean number of PGCs aggregating were observed compared with the controls （P 〈 0.05） while the total PGC number in the right and left gonads at each stage did not change （P 〉 0.05）. Conclusion： The present study provides evidence that E2 has significant effects on the localization of PGCs in male right, but not female left, gonads of chicken embryos at stages 28-30, compared with controls. （Asian J Andro12008 Mar; 10： 243-248）

Unilateral post-chemotherapy robot-assisted retroperitoneal lymph node dissection in Stage II non-seminomatous germ cell tumor:A tertiary care experience

Objective Post-chemotherapy retroperitoneal lymph node dissection(PC-RPLND)represents an integral component of the management of patients with non-seminomatous germ cell tumor(NSGCT).Modified templates have been proposed to minimize the surgical morbidity of the procedure.Moreover,the implementation of robotic surgery in this setting has been explored.We report our experience with unilateral post-chemotherapy robot-assisted retroperitoneal lymph node dissection(PC-rRPLND)for clinical Stages IIA and IIB NSGCTs.Methods A retrospective single institution review was performed including 33 patients undergoing PC-rRPLND for Stages IIA and IIB NSGCTs between January 2015 and February 2019.Following orchiectomy,patients were scheduled for chemotherapy with three cycles of bleomycin-etoposide-cisplatin.Patients with a residual tumor of<5 cm and an ipsilateral metastatic disease on pre-and post-chemotherapy CT scans were eligible for a unilateral template in absence of rising tumor markers.Descriptive statistics were provided for demographics,clinical characteristics,intraoperative and postoperative parameters.Perioperative,oncological,and functional outcomes were recorded.Results Overall,7(21.2%)patients exhibited necrosis or fibrosis;14(42.4%)had mature teratoma;and 12(36.4%)had viable tumor at final histology.The median lymph node size at surgery was 25(interquartile range[IQR]21-36)mm.Median operative time was 180(IQR 165-215)min and no major postoperative complications were observed.Anterograde ejaculation was preserved in 75.8%of patients.Median follow-up was 26(IQR 19-30)months and a total of three recurrences were recorded.Conclusion PC-rRPLND is a reliable and technically reproducible procedure with safe oncological outcomes and acceptable postoperative ejaculatory function in well selected patients with NSGCTs.

Testicular mixed germ cell tumor:A case report

BACKGROUND Testicular mixed germ cell tumors(TMGCTs)are rare malignant tumors that are more common in men aged 20–40 years.TMGCTs comprise two or more types of germ cell tumors that primarily affect the testis.Their onset is undetectable;thus,early diagnosis is challenging.However,early recognition and diagnosis substantially improve patient prognosis.CASE SUMMARY We evaluated a rare case of TMGCT in a male patient presenting with recurrent fever and left supraclavicular lymphadenectasis instead of testicular enlargement and pain,which may easily lead to misdiagnosis.We report the clinical signs and symptoms,histopathological characteristics,and immunohistochemical results of this case of malignant TMGCT.CONCLUSION Our case,which was typical with multiple components,along with a literature review,may serve as a basis for early diagnosis.

刺参germ cell-less基因表达分析及转录因子预测

不同性别的刺参(Apostichopus japonicus)在生长速度、免疫能力等方面具有显著差异,解析其性别决定和性别分化机制具有重要的理论和经济价值。目前,刺参的性腺发育机制尚不清晰,挖掘性腺发育相关基因是解析其发育机制的重要途径。研究表明,germ cell-less基因在哺乳动物性腺发生中起重要作用,但无脊椎动物中关于germ cell-less基因的研究较少。本研究从刺参基因组中鉴定了germ cell-less (Ajgcl)基因片段,随后通过c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)获得其全长c DNA序列。通过荧光定量PCR (real-time quantitative PCR,RT-q PCR)揭示了Ajgcl在成体组织中呈现泛表达状态,性腺中表达量最高,且雌性性腺中的表达量是雄性性腺中表达量的2.25倍。随着卵子的发生,Ajgcl的表达量呈现先上升后下降的动态表达变化,而在精子发生的过程中其表达量变化不大,意味着其可能在卵子发生过程中发挥重要作用。在整个胚胎发育过程中均能检测到Ajgcl转录本,意味着Ajgcl作为母源因子可能与原始生殖细胞的形成有关。此外,Ajgcl基因启动子中具有Oct-1、FOXD3、PAX-6、CRP、c-Myb和NF-1等转录因子的结合位点。本研究为深入解析germ cell-less基因在刺参等无脊椎动物性腺发育中发挥的功能奠定了基础。

Primordial germ cell-mediated transgenesis and genome editing in birds

Transgenesis and genome editing in birds are based on a unique germline transmission system using primordial germ cells(PGCs), which is quite different from the mammalian transgenic and genome editing system. PGCs are progenitor cells of gametes that can deliver genetic information to the next generation. Since avian PGCs were first discovered in nineteenth century, there have been numerous efforts to reveal their origin, specification, and unique migration pattern, and to improve germline transmission efficiency. Recent advances in the isolation and in vitro culture of avian PGCs with genetic manipulation and genome editing tools enable the development of valuable avian models that were unavailable before. However, many challenges remain in the production of transgenic and genome-edited birds,including the precise control of germline transmission, introduction of exogenous genes, and genome editing in PGCs.Therefore, establishing reliable germline-competent PGCs and applying precise genome editing systems are critical current issues in the production of avian models. Here, we introduce a historical overview of avian PGCs and their application, including improved techniques and methodologies in the production of transgenic and genome-edited birds, and we discuss the future potential applications of transgenic and genome-edited birds to provide opportunities and benefits for humans.

Improved Isolation and Culture of Embryonic Germ Cells from Guanzhong Dairy Goat

A total of 219 embryonic-germ-cell-like （EG-like） clumps were derived from 15 selected goat fetuses. Isolation of primordial germ cells （PGCs） based on co-culture with primary goat embryonic fibroblast showed no difference from traditional feeder layer-based culture method used in mouse and human. The putative primary EG colonies were multilayer clumps of compact cells with unclear cell-cell boundaries. Three subculture methods of goat EG-like colony, traditional enzymatic digestion, mechanical cutting and combination of the both, were compared in this study. As a result, EG-like colonies traditionally disassociated with collagenase 1V could be subcultured for up to 4 passages. And the mechanically disaggregated EG-like colonies were successfully maintained 9-12 passages with or without enzymatic treatment. The pluripotency of the EG-like colonies was identified by their specific marker staining, spontaneous differentiation and embryoid bodies （EBs） formation in vitro. Most goat EG-like colonies （〉 80%） were AKP positive and immunocytochemically characterized with positive SSEA-1, Oct-4 and c-kit staining but SSEA-4. Under the condition of delaying passage, goat EG-like cells could differentiate into fibroblast-like, epithelium-like, and neuron-like cells. In addition, EBs could be obtained successfully in routine hanging drop culture. The serum free culture system （feeder layer-based） used in this study was suitable for keeping PGCs and EG-like cells in their undifferentiated condition, but failed to converse them to immortal cells. These results indicated that mechanical cutting is an effective method for passaging goat EG cell colonies. However, the microenvironment of conversing EG cells to immortal cells is still unclear.

Molecular and epigenetic pathogenesis of germ cell tumors

The development of germ cell tumors(GCTs)is a unique pathogenesis occurring at an early developmental stage during specification,migration or colonization of primordial germ cells(PGCs)in the genital ridge.Since driver mutations could not be identified so far,the involvement of the epigenetic machinery during the pathogenesis seems to play a crucial role.Currently,it is investigated whether epigenetic modifications occurring between the omnipotent two-cell stage and the pluripotent implanting PGCs might result in disturbances eventually leading to GCTs.Although progress in understanding epigenetic mechanisms during PGC development is ongoing,little is known about the complete picture of its involvement during GCT development and eventual classification into clinical subtypes.This review will shed light into the current knowledge of the complex epigenetic and molecular contribution during pathogenesis of GCTs by emphasizing on early developmental stages until arrival of late PGCs in the gonads.We questioned how misguided migrating and/or colonizing PGCs develop to either type Ⅰ or type Ⅱ GCTs.Additionally,we asked how pluripotency can be regulated during PGC development and which epigenetic changes contribute to GCT pathogenesis.We propose that SOX2 and SOX17 determine either embryonic stem cell-like(embryonal carcinoma)or PGC-like cell fate(seminoma).Finally,we suggest that factors secreted by the microenvironment,i.e.BMPs and BMP inhibiting molecules,dictate the fate decision of germ cell neoplasia in situ(into seminoma and embryonal carcinoma)and seminomas(into embryonal carcinoma or extraembryonic lineage),indicating an important role of the microenvironment on GCT plasticity.

Surgical treatment of metastatic germ cell cancer

Among young men between the ages of 15 and 40 years,germ cell cancer is the most common solid tumor[1].The worldwide incidence of germ cell cancer is 70000 cases.Compared to all solid tumors of men,germ cell cancer accounts for 1%of all male tumors.Nevertheless,the mortality of this rare tumor entity is about 13%since 9507 patients died worldwide of germ cell cancer.The improvement in survival of germ cell cancer patients is due to a multimodal treatment of germ cell cancer including cisplatin-based chemotherapy and surgery leading to higher cure-rates even in advanced stages[1],whereas the increasing incidence of germ cell cancers cannot be thoroughly explained.In this article we review the current indications for surgery in metastatic germ cell cancers,highlight the strength and weaknesses of techniques and indications and raise the question how to improve surgical treatment in metastatic germ cell cancer.

Germ cell apoptosis induced by Ureaplasma urealyticum infection

Aim: To study the effect of Ureaplasma urealyticum (UU) infection on germ cell apoptosis of male rats. Meth-ods: Male rats were infected artificially with UU serotype 8 (T_(960)). Morphological changes of germ cells in the sem-iniferous tubules and the lumen of the epididymides were observed under the light microscope. Fluorescence-conjugatedpolyclonal antibodies to Fas and Fas ligand (FasL) were used to localize Fas and FasL. TUNEL staining of germ cellsand Sertoli cells was performed by the AKPase method. TUNEL-positive rate (% positive cells) and TUNEL-positivearea (area occupied by stained cells) were analysed by KS400 Image Analysis System. The DNA laddering analysiswas performed by agarose gels electrophoresis. Results: In those rats infected with UU; (1) Exfoliated germ cellswere dramatically increased. Many multinucleated giant cells were found in the seminiferous tubules and the lumen ofthe epididymides. (2) The number of TUNEL-positive cells and the TUNEL-positive area were significantly increased.(3) The expression of Fas and FasL in germ cells and Sertoli cells was up-regulated. (4) Discrete bands of fragmentedDNA were found in the testicular cells. Conclusion: In male rats, germ cell apoptosis was increased in UU infec-tion . (Asian J Androl 2001 Sep; 3: 199 - 204)

Generation of male germ cells from induced pluripotent stem cells （iPS cells）： an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem （iPS） cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitrodifferentiation and in vivotransplantation. Embryoid bodies （EBs） were obtained from iPS cells using leukaemia inhibitor factor （LIF）-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in StraSand Vasa mRNA in the EBs derived from iPS cells, iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells （SSCs）, as evidenced by their expression of VASA, as well as CDH1 and GFRal, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

Assessment of germ cell apoptosis in cryptorchid rats

Aim: To investigate the relationship between germ cell degeneration and apoptosis in cryptorchid rats. Methods: Thirteen 21-day-old Wistar rats were made unilaterally cryptorchid by closing the left inguinal canal. At day 30 (Group 1, n=6) and day 60 (Group 2, n=7) after operation, the testes were removed for histopathological examination. The controls (n=8) were sham operated and were sacrificed at day 60. Germ cell apoptosis was assessed by means of the TUNEL method. Results: Spermatogenesis was arrested and the testicular and seminiferous tubular diameters were significantly reduced In the unilateral undescended testes (UUTs) compared with the contralateral descended testes (CDTs) and the control rats. However, atrophic changes, pathological calcification, necrosis of seminiferous tubule, and absence or sloughing of germ cells were not found in all the animals. The spermatocytes were the main type of germ cells undergoing apoptosis in all the groups. In the UUTs, there was a significant and time-dependent increase in the mean apoptotic index. By 60 days after surgery, increased apoptosis in germ cells was also observed in the CDTs. Conclusion: Apoptosis is the predominant mechanism of germ cell death rather than atrophy and necrosis in cryptorchidism.

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period

Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immun-ofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary sperma-tocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion: GCNF may play important roles in spermatogenesis, capacitation and fertilization.

Effect of prolonged cryptorchidism on germ cell apoptosisand testicular sperm count

Aim: To evaluate the long term effect of experimental cryptorchidism on germ cell apoptotic rate and testicular sperm content in adult rats. Methods: Bilateral cryptorchidism was created in 40 adult male Sprague-Dawley rats by surgically manipulating the testes into the abdominal cavity and closing the internal inguinal ring. The rats were sacrificed and the testes removed 6 hours and 2, 4, 7, 21, 28 and 56 days after cryptorchidism. Germ cell apoptosis was quantified by means of TUNEL assay and apoptosis was confirmed using transmission electron microscopy. Results: The rate of apoptosis peaked at 4 days of cryptorchidism and then progressively declined to a nadir at 14 days of cryptorchidism. At 56 days of cryptorchidism, the germinal epithelium was largely depleted by the apoptotic process and only a few mature sperm were seen within the testis. At this point, a few tubules were seen to be repopulating with primary spermatocytes and the level of germ cell apoptosis began to increase marginally. Testicular sperm count (TSC) began to decline rapidly at day 7 of cryptorchidism. Only a few mature sperm were found in the testes of rats following 56 days of cryptorchidism. Multinucleated giant cells (MGC) were most numerous within the seminiferous tubules at day 4. At day 7, 35 % of MGCs were TUNEL positive. At all subsequent time points, however, MGCs fail to stain positive for apoptosis. This resumption of increased apoptosis coincided with the appearance of a population of primary spermatocytes in some seminiferous tubules. Moreover, there was not a corresponding increase in the number of mature sperm after 56 days of cryptorchidism. Conclusion: The decline in germ cell apoptosis after 4 days of cryptorchidism can be attributed to be the result of an overall depletion of germ cells. It appears that after a prolonged cryptorchidism (56 days), there is a limited resumption of spermatogenesis presumably as a result of a decrease in the maturing germ cells undergoing programmed cell death.