Guinea Pig Testes Research Articles

Spermatogenesis after Transplantation of Adipose Tissue-Derived Stem Cells in Azoospermic Guinea Pigs: A Histological and Histomorphometric Study

Background: The purpose of this research was to determine histomorphometric changes in busulfan-induced azoospermia after transplantation of AdSCs in guinea pig. AdSCs were isolated from adipose tissue around the testes of guinea pigs and characterized for mesenchymal properties. Materials and Methods: Guinea pigs were allocated into three groups, including the control group without any intervention. To induce azoospermia, groups 2 and 3 received a dose of 40 mg/kg of busulfan with 21 days interval. Group 3 received 1×106 AdSCs in their seminiferous tubules of left testes, 35 days following last busulfan injection, while right testis in the group was considered for comparison as controls. Sixty days following transplantation of cell, histomorphometric and histopathologic changes of the experiments were assessed. Results: After AdSCs’ transplantation, normal spermatogenesis appearance was noticed compared to busulfan-induced azoospermia and AdSCs recovered spermatogenesis, and our findings can be added to the literature in treating azoospermic infertilities. Conclusion: The transplanted AdSCs could induce production of germinal cells using testicular seminiferous tubules and were an effective source in treating azoospermia.[GMJ.2018;7:e1000]

Morphological and Biochemical Evaluation of the Testes of Hyperglycemic Guinea Pigs Treated with Cyperus Esculentus Extract

In this study, we aimed to evaluate the effects of Cyperus esculentus extract on Testicular Morphology and Biochemistry post streptozotocin-treatment in Male Guinea Pigs. Twenty guinea pigs were divided into four groups containing five pigs each. Group A served as the negative control was treated with 5ml/kg body weight of normal saline orally, Group B were treated with a single dose of 65mg/kg body weight of streptozotocin (STZ) intraperitoneally, Group C were treated with a single dose of 65mg/kg body weight of STZ and daily treatment with 150mg/kg of CEE orally and Group D were treated with a single dose of 65mg/kg body weight of STZ and daily treatment with 150mg/kg of CEE orally for 20days. Morphological scores and biochemical evaluation were done. Results showed a significant decrease in testicular weight, number of spematogonia, Leydig cells and Sertoli cells, levels of SOD and CAT as well as a significant increase in the level of MDA after the administration of STZ alone when compared to the control. A significant decrease in the levels of testosterone and FSH as well as a decrease in the level of LH was observed in group B when compared to the control. However, the groups that were co-treated with the extract had a better parameters and histological profiles when compared to the positive control group. CEE has a dose-dependent ameliorative effect on STZ-induced testicular hormonal, oxidative and histomorphometric degeneration.

Studies on Postnatal Development of Testis of Guinea Pig (Cavia porcellus)

The histology and histochemistry of the testis of guinea pig of various postnatal age groups was conducted. A total of 24 guinea pigs of four different postnatal ages with six male animals each were collected from the Department of Laboratory Animal Medicine, Madhavaram Milk Colony, Chennai as per the Ethical committee approval. After collection, animals were euthanized as per CPCSEA norms and testis was dissected out and was cut into small pieces, fixed and processed for paraffin embedding. Sections of 4–5 µm thickness were cut and used for the routine and special histological and histochemical staining techniques. Testes wereencapsulated by tunica vaginalis and tunica albuginea. Septa from the capsule divided the testicular parenchyma into lobules. Each lobule consisted of seminiferous tubules which consisted of spermatogenic cells in stratified layers and sertoli cells. Pre-weaning and weaning group of guinea pigs seminiferous tubules showed wide lumen with only type 1 and type 2 spermatogonia and sertoli cells. Young and adult animals seminiferous tubules showed narrow lumen with type 1 and type 2 spermatogonia, primary spermatocytes in various stages of differentiation, secondary spermatocytes, spermatids (early and late) and sertoli cells. Sertoli cells were large oval shaped cells with lightly stained irregular shaped nucleus. Interstitial tissue contained leydig cells in all ages.Leydig cells appeared as varied in shape. In all the age groups studied, PAS activity was noticed in the capsule and basement membrane. The micrometrical parameters increased as age advanced in both right and left testis.

Drinking Non-nutritive Sweetness Solution of Sodium Saccharin or Rebaudioside a for Guinea Pigs: Influence on Histologic Change and Expression of Sweet Taste Receptors in Testis and Epididymis.

Saccharin sodium and rebaudioside A are extensively used as non-nutritive sweeteners (NNSs) in daily life. NNSs elicit a multitude of endocrine influences on animals, differing across species and chemically distinct sweeteners, whose exposure induce activation of sweet taste receptors in oral and extra-oral tissues with consequences of metabolic changes. To evaluate the influence of NNSs on histologic change and expression of sweet taste receptors in testis and epididymis of young male guinea pigs, thirty 4-week-old male guinea pigs with body weight 245.73 ± 6.02 g were randomly divided into five groups (n = 6) and received normal water (control group) and equivalent sweetness low dose or high dose of sodium saccharin (L-SS, 1.5 mM or H-SS, 7.5 mM) or rebaudioside A (L-RA, 0.5 mM or H-RA, 2.5 mM) solution for 28 consecutive days. The results showed that the relative testis weight in male guinea pig with age of 56 days represented no significant difference among all groups; in spite of heavier body weight in L-SS and H-RA, NNS contributes no significant influence on serum testosterone and estradiol level. Low-dose 0.5 mM rebaudioside A enhanced testicular and epididymal functions by elevating the expressions of taste receptor 1 subunit 2 (T1R2) and gustducin α-subunit (GNAT3), and high-dose 7.5 mM sodium saccharin exerted adverse morphologic influences on testis and epididymis with no effect on the expression of T1R2, taste receptor 1 subunit 2 (T1R3), and GNAT3. In conclusion, these findings suggest that a high dose of sodium saccharin has potential adverse biologic effects on the testes and epididymis, while rebaudioside A is a potential steroidogenic sweetener for enhancing reproductive functions.

Spermatogenesis after Transplantation of Adipose Tissue-Derived Stem Cells in Azoospermic Guinea Pigs: A Histological and Histomorphometric Study.

Background: The purpose of this research was to determine histomorphometric changes in busulfan-induced azoospermia after transplantation of Adipose Tissue-Derived Stem Cells (AdSCs) in guinea pig. AdSCs were isolated from adipose tissue around the testes of guinea pigs and characterized for mesenchymal properties. Materials and Methods: Guinea pigs were allocated into three groups, including the control group without any intervention. To induce azoospermia, groups 2 and 3 received a dose of 40 mg/kg of busulfan with 21 days interval. Group 3 received 1×106 AdSCs in their seminiferous tubules of left testes, 35 days following last busulfan injection, while right testis in the group was considered for comparison as controls. Sixty days following transplantation of cell, histomorphometric and histopathologic changes of the experiments were assessed. Results: After AdSCs’ transplantation, normal spermatogenesis appearance was noticed compared to busulfan-induced azoospermia and AdSCs recovered spermatogenesis, and our findings can be added to the literature in treating azoospermic infertilities. Conclusion: The transplanted AdSCs could induce production of germinal cells using testicular seminiferous tubules and were an effective source in treating azoospermia

Spermatogenesis after Transplantation of Adipose Tissue-Derived Stem Cells in Azoospermic Guinea Pigs: A Histological and Histomorphometric Study

Background: The purpose of this research was to determine histomorphometric changes in busulfan-induced azoospermia after transplantation of AdSCs in guinea pig. AdSCs were isolated from adipose tissue around the testes of guinea pigs and characterized for mesenchymal properties.Materials and Methods: Guinea pigs were allocated into three groups, including the control group without any intervention. To induce azoospermia, groups 2 and 3 received a dose of 40 mg/kg of busulfan with 21 days interval. Group 3 received 1×106 AdSCs in their seminiferous tubules of left testes, 35 days following last busulfan injection, while right testis in the group was considered for comparison as controls. Sixty days following transplantation of cell, histomorphometric and histopathologic changes of the experiments were assessed.Results: After AdSCs’ transplantation, normal spermatogenesis appearance was noticed compared to busulfan-induced azoospermia and AdSCs recovered spermatogenesis, and our findings can be added to the literature in treating azoospermic infertilities.Conclusion: The transplanted AdSCs could induce production of germinal cells using testicular seminiferous tubules and were an effective source in treating azoospermia.[GMJ.2018;7:e1000]

Morphological development of the testicles and spermatogenesis in guinea pigs (Cavia porcellus Linnaeus, 1758)

Abstract Introduction: Understanding the dynamics of spermatogenesis is crucial to clinical andrology and to understanding the processes which define the ability to produce sperm. However, the entire process cannot be modeled in vitro and guinea pig may be an alternative as animal model for studying human reproduction. Objective: In order to establish morphological patterns of the testicular development and spermatogenesis in guinea pigs, we examined testis to assess changes in the testis architecture, transition time from spermatocytes to elongated spermatids and stablishment of puberty. Materials and methods: We used macroscopic analysis, microstructural analysis and absolute measures of seminiferous tubules by light microscopy in fifty-five guinea pigs from one to eleven weeks of age. Results: Differences in relation to mass, length and width of the testes appeared at week 3 and were intensified in week 6 with the puberty. At week 2 the first spermatocytes at preleptotene/leptotene appeared, indicating the onset of meiosis. At week 6 the seminiferous tubules formed lumen, differentiated Sertoli cells and all kinds of germ cells; spermatozoa released into the lumen and the largest expansion of tubular diameter and height of the seminiferous epithelium occurred. Transition time from spermatocytes to elongated spermatids was estimated in 21 days. Conclusion: We conclude that the testicular development and puberty are faster than in other mammal and rodent species, demonstrating the suitability of the guinea pig testes as a model for studies of the testicular development and spermatogenesis.

Effects of fluoride and aluminum on expressions of StAR and P450scc of related steroidogenesis in guinea pigs' testis.

A lot of studies have shown that fluoride and aluminum have toxic effect on male reproductive system, but the mechanism of which and the interaction between fluoride and aluminum is still unknown. This study investigated the effects of fluoride (NaF) or/and aluminum (AlCl3) on serum testosterone level, gene and protein expression levels of Steroidogenic Acute Regulatory Protein (StAR) and Cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) in the testes of guinea pigs. Fifty-two guinea pigs were divided randomly into four groups (Control, HiF, HiAl and HiF + HiAl). Fluoride (150 mg NaF/L) or/and aluminum (300 mg AlCl3/L) were orally administrated to male guinea pigs for 13 weeks. The results showed that F and Al reduced number and elevated abnormal ratio of sperm. Meanwhile, the concentrations of serum testosterone in all experimental groups were decreased. P450scc protein expression was significantly reduced in all treatment groups, and StAR expression was decreased remarkably in HiF group and HiF + HiAl group. The levels of StAR mRNA in three groups were reduced by 53.9%, 21.4% and 33.4%, respectively, while the expressions of P450scc mRNA were reduced by 67.8%, 17.0% and 47.8%. Therefore, we concluded that F induced the reduction in testosterone and sperm amount, and thus in lower fertility, which might occur as a consequence of depressed StAR and P450scc mRNA expression. There were no synergistic effects between F and Al, instead, Al weakened the toxicity of F to some extents. The results indicated that Al had antagonism effects on F.

High‐purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production.

Panax ginseng Improves Senile Testicular Function in Rats

We reported previously that the administration of Korean red ginseng water extract (KRG-WE) protected the guinea pig testis against damage induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (a potent endocrine disruptor). We also found that crude saponin from ginseng was the active ingredient responsible for this protection. Here, we examined the biological role of KRGWE in an animal model of age-induced dysfunction of spermatogenesis. Twenty-four male Sprague-Dawley (six 2-monthold and eighteen 12-month-old) rats were used. The young and old control groups received only vehicle. The ginseng saponin (GS)- and KRG-WE-treated groups received GS (40 ㎎/㎏ body weight/day) and KRG-WE (200 ㎎/㎏ body weight/day), respectively, for 4 months. The number of cells, Sertoli cell index, Johnsen’ score, and sex hormone levels decreased significantly with age. However, the administration of KRG-WE and GS markedly improved the number of germ cells, seminiferous tubular size, and Johnsen’ score in the old rats. Ginseng produced a distinct testicular histological improvement in old rats. KRG-WE and GS elevated testosterone levels, while attenuating the aberrant increase in follicle stimulating hormone and luteinizing hormone levels. Sperm kinematics evaluated by a computer-assisted sperm analyzer demonstrated improvement in the percentage of motile sperm, progressive sperm motility, and curvilinear velocity associated with sperm quality, supporting the beneficial role of red ginseng in senile spermatogenesis. Overall, the total water extract had a more potent effect than the corresponding saponin fraction. In conclusion, Korean red ginseng rejuvenated age-induced testicular dysfunction. Additionally, the total water extract was more potent than the corresponding saponin fraction.

On the different lipolytic capability of diverse organs from young adult guinea pigs. A chromatographic study

In this study we conducted in-vitro incubation of whole-tissue homogenate of young adult guinea pig heart, kidney, testis, and spleen at pH 7.4 and 38°C for 60 min. Subsequent TLC analysis revealed: (a) a noticeably high level of MLCL and a very low level of CL in control testis, indicating possible in-vivo deacylation of CL (b) a high level of CL in control guinea pig heart and kidney samples, and no MLCL detected (c) MLCL was only produced (in both heart and kidney) subsequent to in-vitro incubation (d) a very low level of CL was present in control samples of guinea pig spleen and no MLCL was produced subsequent to in-vitro incubation (e) guinea pig heart contains both ethanolamine (PE) and choline plasmalogens (PC) (f) PE plasmalogen is the only alkenyl species present in guinea pig kidney, testes, and spleen (g) sphingomyelin (SM) is uniquely higher in kidney than testis and spleen and very low in the heart (h) subsequent to in-vitro incubation, ceramide is produced in the kidney and testes only, with...

Comparative Testicular Histopathological Effects of Artemisinin Derivatives and Some ACTs in the Guinea-pig

Artesunate and dihydroartemisinin are artemisinin derivatives, which are effective antimalarial agents used in the treatment of malaria. Combination of artemisinins and other standard antimalarial drugs (ACTs) have resulted in better cure rates of Plasmodium infections. In this study, the histolopathological effects of half, normal and double clinical doses of artesunate, dihydroartemisinin, artesunate/amodiaquine, artesunate/sulfadoxine/pyrimethamine and artemether/lumefantrine on the testes of guinea-pigs were investigated. Mean testicular weight and circumference were both significantly (p

Effects of Vanadium Pentoxide on the Histological and Sperm Parameters of Male Guinea Pigs

The pharmacological effects of intraperitoneal administration of different doses of vanadium pentoxide (V2O5) on the histological and sperm parameters of male guinea pigs were investigated. Also investigatedwere the effects of oral pretreatment with different doses of vitamin E (a known protein kinase C inhibitor) on the V2O5 -induced responses of the testis and liver of male guinea pigs. In n = 5 experiments, vanadium pentoxide in the dose range of 4.5-12.5mg/kg caused destruction of the testicular and liver architecture. This was characterized by areduction in spermatogonia, destruction of seminiferous tubules, necrosis of the testicular tissues, necrosis of liver cells, fatty cells infiltration and vacoulation. Oral administration/ pretreatment with vitamin E in the dose range of 500-2000I.U caused a reversal of the vanadium pentoxide –induced histological damages of the testis and the liver cells. Furthermore, in n = 5 animals experiments, Vanadium pentoxide (4.5-12.5 mg/kg/) caused a statistically significant increase in the percentage basal cell death, from 5.0 to 75.0 ± 1.0%, reduction in sperm motility from 90.0 to 31.0 ± 3.9%, reduction in sperm count from 80.0 x106cells/ml to 25.0± 4.0 x 106cells/ml and alteration in the spermatic cell morphology ( i.e. causing a change in the cellular structure of sperm cells and an increase in abnormal cells count) of the male guinea pigs. These inhibitory effects were significant at P < 0.05 (ANOVA). These effects were all dose- and time-dependent and may have a role in oxidative pathology of vanadium pentoxide.

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ-cell morphology remain unclear. This study evaluates the short-term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30-day old) and adult (65- and 135-day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non-steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post-pubertal and adult guinea pigs, in addition to causing germ-cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.

Immunologic and testicular response in guinea pigs after unilateral traumatic orchitis.

An acute and intense trauma was inflicted in one of the testes of adult guinea pigs. The immunological response and histological changes developed both in the injured and contralateral glands were studied at different intervals of time. It was observed: 1) Necrotic seminiferous tubules and a granulomatous cell reaction were seen in the injured gonad at 15 days. This was followed by a reduction of the mononuclear cell infiltration at 48 days. Finally a fibrotic tissue replaced the damaged areas at around 100 days. 2) Foci showing the classical picture of immunologic orchitis appeared in the contralateral gland at 15 and 48 days. Less frequent lesions were detected in the final period of the experiment. 3) These lesions were accompanied by cellular immunity detectable by the macrophage migration inhibition test at 15 and 48 days. With the exception of cytophilic activity also detected in same periods, no antibodies of hemagglutinating and sperm-agglutinating types could be found.

Detection of type 2 herpes simplex virus in cells of spermatogenic epithelium in infected testes of guinea pigs

We developed a model of herpetic orchitis in guinea pigs. Intratesticular inoculation of type 2 herpes simplex virus suspension results in infection of the testicular spermatocytes and spermatides. The possibility of viral infection dissemination from infected into intact testis is proven.

The Effects of Mainstream and Sidestream Cigarette Smoke on Sperm Production: Role Retinoic Acid

Vitamin A is an important signaling molecule for testicular cell growth and differentiation. Our preliminary studies have indicated that cigarette smoke (CS) causes a disturbance in retinoid metabolism and signaling in lung. However, nothing is known on the effects of smoking on retinoids in testis. In this study, we determined the effects of mainstream (MS) and sidestream (SS) smoke not only on sperm quantity and motility, but also on the levels of retinoids in the testis of guinea pigs in the presence or absence of retinoic acid (RA) exposure. Male guinea pigs (4 wk-old) were exposed to MS, SS or sham (SH) smoke twice daily from 3 cigarettes for 6 weeks. Once a day before CS exposure, half of the animals received RA via direct inhalation, while others were kept as room controls (RC). After 6 weeks of smoke exposure, testes were extracted and analyzed by HPLC to determine the levels of retinoids. CS altered sperm motility (MS<SS<SH<RC) and sperm count (RC=SH<MS<SS), respectively, whereas treatment with RA had no effect on sperm count and motility. Both MS and SS smoking caused a significant decrease in 13-cis, 9-cis and all-trans RA in testis whereas retinol and retinyl esters were increased. Our results indicate that CS not only affects sperm production, sperm motility but also retinoid levels. (Supported by R25 GM064319 and DAMD 17-03-2-0054).

Mono-(2-ethylhexyl) Phthalate (MEHP) Induces Spermatogenic Cell Apoptosis in Guinea Pig Testes at Prepubertal Stage In Vitro

The effects of mono-(2-ethylhexyl) phthalate (MEHP), an active metabolite of di-(2-ethylhexyl) phthalate (DEHP), on prepubertal guinea pig testes in vitro were investigated. The testes of 35-day-old guinea pigs were surgically excised. They were seeded in a defined medium containing antibiotics and administered MEHP at concentrations of 1, 10, and 100 nmol/ml, respectively. The control groups were administered a similar volume of corn oil vehicle. The tissues were incubated for 3, 6, and 9 h. The specimens were collected at 3, 6, and 9 h after treatment. They were fixed in 4% paraformaldehyde or 5% glutaraldehyde. For quantitation of the apoptotic spermatogenic cells, the terminal dUTP nick end-labeling (TUNEL) staining was performed by light microscopy. Detachment and displacement of spermatogenic cells, thin seminiferous epithelia, and Sertoli cell vacuolization were observed. Maximal testicular damage was recognized at 100 nmol/ml and 9 h after MEHP treatment. The percentage (%) of apoptotic spermatogenic cells significantly increased at 3, 6, and 9 h after treatment, compared to the control groups. Because the loss of spermatogenic cells by MEHP treatment varies among species, the present study, using guinea pigs, was designed and conducted to obtain further information.

Effects of Nitric Oxide on the Induction of Experimental Allergic Orchitis in Guinea Pig

Background: Production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathology of autoimmune disease. It is unknown whether iNOS expression is increased within testes and whether iNOS and NO have essential roles in the pathogenesis of EAO. Methods: EAO was induced in guinea pig testes at 17 days after secondary immunization by administration of crude extract (CE) and purified glycoprotein 1 (GP1) from normal guinea pig testes. iNOS gene expression was assessed by RT-PCR and Northern blot analysis in testes. Localization of iNOS and Mac-1 and the indicator of NO-mediated tissue injury, nitrotyrosine, were detected in the testicular lesion by immunohistochemistry. Results: In control testes, inflammation and iNOS gene expression were not detected, whereas, in CE- and GP1-injected testes, inflammation and marked iNOS gene expression were evident at day 17 after secondary immunization. Immunohistochemistry of Mac-1 showed the colocalization with iNOS protein and nitrotyrosyl proteins in intertubules, suggesting that NO produced by infiltrated macrophages may be involved in inflammatory lesions of intertubules. Intraperitoneal administration of aminoguanidine significantly prevented EAO with reduction of inflammation, iNOS expression and nitrotyrosine formation. Conclusion: These results suggest that NO production by macrophages may be important in the pathogenesis of CE- and GP1-induced EAO. Furthermore, this study demonstrated the therapeutic potential of iNOS inhibitor in the treatment of inflammatory and autoimmune mediated-diseases.

Monoclonal antibody G3 epitope location on Guinea pig sperm membrane protein, sperad.

A protein isolated from guinea pig testes was purified, characterized as having a molecular weight of 34 kDa, and subjected to amino acid sequencing. A monoclonal antibody to the protein (G11) was found to cross-react with a second monoclonal antibody (G3). Two peptide sequences derived from the purified protein, labeled as 34 kDa G11, show sequence homology with sperad. Sperad is a transmembrane protein present in the peri-acrosomal plasma membrane of guinea pig spermatozoa. Both G11 and G3 monoclonal antibodies recognise antigens on the equatorial segment plasma membrane of guinea pig spermatozoa following the acrosome-reaction. Therefore, experiments were designed to check whether monoclonal antibody G3 epitope is present on the sperad. The intra-cytoplasmic domain and the extra-cytoplasmic domain of sperad was amplified from a guinea pig testes cDNA expression library by polymerase chain reaction and cloned into pGEM T-Easy vector. The recombinant pGEM T-Easy plasmids were subjected to in vitro transcription and translation by rabbit reticulocyte lysate system. The resulting translated products were immunoprecipitated with monoclonal antibody G3. The results obtained from this study confirmed that monoclonal antibody G3 epitope is located on the extra-cytoplasmic domain but not on the intra-cytoplasmic domain of sperad.