Indian Journal of Experimental Biology


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ISSN : 0019-5189

            CODEN : IJEB (A6) 43(11)                                 933-1112 (2005)

VOLUME 43

                  NUMBER 10

November 2005

 

 

 

CONTENTS

 

Special issue on Male Reproductive Biology

 

Review Articles
Leydig cells, thyroid hormones and steroidogenesis

939

      S M L Chamindrani Mendis-Handagama & H B Siril Ariyaratne

 

 

 

Mechanism, measurement and prevention of oxidative stress in male reproductive physiology

963

      Ashok Agarwal & Sushil A Prabakaran

 

 

 

A treatise on hazards of endocrine disruptors and tool to evaluate them

975

      Partha Roy & Ben M J Pereira

 

 

 

FSH, the neglected sibling: Evidence for its role in regulation of spermatogenesis and Leydig cell function

993

      V Sriraman & A J Rao

 

 

 

Proteins implicated in sperm capacitation

1001

      Kasturi Mitra & S Shivaji

 

 

 

Does human sperm nuclear DNA integrity affect embryo quality?

1016

      Silpi Acharyya, Sagarika Kanjilal & Asok K Bhattacharyya

 

 

 

Current trends in evaluation of sperm function: In vitro selection and manipulation of male gametes for assisted conception

1023

      Alex C Varghese, Barnali Sinha & Asok K Bhattacharyya

 

 

 

Male contraception: Expanding reproductive choice

1032

      M Rajalakshmi

 

 

 

Perspectives of contraceptive choices for men

1042

      N K Lohiya, B Manivannan, S S Bhande, S Panneerdoss & Shipra Garg

 

 

 

Papers

 

Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation

1048

      Amiya P Sinha Hikim, Yanira Vera, Rashid i Elhag, Yanhe Lue,

      Yu-Gui Cui, Vanisha Pope, Andrew Leung, Vince Atienza, 

       Christina Wang & Ronald S Swerdloff

Experimental hyperthyroidism-induced oxidative stress and impairment of       antioxidant defence system in rat testis

1058

      Dipak K Sahoo, Anita Roy, Shravani Bhanja & G B N Chainy

 

 

 

Effect of estradiol on expression of cytoskeletal proteins during spermatogenesis in testis of sexually mature rats

1068

      J Choudhuri, M Aleem, V Padwal, P Das Gupta, R D’ Souza, S Pathak, N Balasinor & M K Gill-Sharma

 

 

 

Germ cell death and their removal during intial stages of testicular ischemia and cryptorchidism: A comparative analysis

1080

      M M Misro, S P Chaki & Dinesh K Gautam

 

 

 

Male infertility: Y-chromosome deletion and testicular aetiology in cases of

      azoo-/oligospermia

1088

      Kiran Singh & Rajiva Raman

 

 

 

Effect of aqueous leaf extract of Azadirachta indica on the reproductive organs

      in male mice

1093

      Raghav Kumar Mishra & Shio Kumar Singh

 

 

Sperm motility inhibiting activity of a phytosterol from Alstonia macrophylla Wall ex A. DC. leaf extract: A tribal medicine

1104

      Debprasad Chattopadhyay, Sandhya R Dungdung, Kaushik Das, Sudipta Saha, Asit B Mandal & Gopal C Majumder

 

 

 

Announcements

 

National Symposium on Biodiversity and Insect Pest Management;

 

National Conference on Aquatic Resources, Aquaculture and Aquashow

(CONFAQUA 2006)

1110

 

 

Author Index

 

Ariyaratne H B Siril

939

Mendis-Handagama & S M L Chamindrani

939

Agarwal Ashok l

963

Prabakaran Sushil A

963

Pereira Ben M J

975

Roy Partha

975

Rao A J

993

Sriraman V

993

Mitra Kasturi

1001

Shivaji S

1001

Acharyya Silpi

1016

Kanjilal Sagarika

1016

Sinha Barnali

1023

Varghese Alex C

1023

Rajalakshmi M

1032

Bhande S S

1042

Garg Shipra

1042

Lohiya N K

1042

Manivannan B

1042

Panneerdoss S

1042

Atienza Vince

1048

Cui Yu-Gui

1048

Elhag Rashid i

1048

Leung Andrew

1048

Lue Yanhe

1048

Pope Vanisha

1048

Sinha Hikim Amiya P

1048

Swerdloff Ronald S

1048

Vera Yanira

1048

Wang Christina

1048

Bhanja Shravani

1058

Chainy G B N

1058

Roy Anita

1058

Sahoo Dipak K

1058

Aleem M

1068

Balasinor N

1068

Choudhuri J

1068

D’ Souza R

1068

Das Gupta P

1068

Gill-Sharma M K

1068

Padwal V

1068

Pathak S

1068

Chaki S P

1080

Gautam Dinesh K

1080

Misro M M

1080

Raman Rajiva

1088

Singh Kiran

1088

Mishra Raghav Kumar

1093

Singh Shio Kumar

1093

Chattopadhyay Debprasad

1104

Das Kaushik

1104

Dungdung Sandhya R

1104

Majumder Gopal C

1104

Mandal Asit B

1104

Saha Sudipta

1104

Bhattacharyya Asok K 1016

Bhattacharyya Asok K

1023

 

 

Keyword index

 

Aging

939

Steroidogenesis

939

Thyroid hormone

939

Male fertility

963

Endocrine disruption

975

Phytoestrogen

975

Reproduction

975

Steroid receptors

975

Xenobiotics

975

Contraception

993

FSH

993

Capacitation

1001

Protein implication

1001

DNA

1016

Embryo

1016

Human sperm

1016

Nuclear DNA

1016

Assisted conception

1023

Male gamete

1023

Sperm function

1023

Androgens

1032

Condom

1032

Progestogens

1032

Carica papaya

1042

Herbal methods

1042

Hormonal methods

1042

RISUG

1042

Vas based methods

1042

Acyline

1048

Apoptosis

1048

Flutamide

1048

Antioxidant

1058

Defences

1058

Mitochondria

1058

Rat

1058

T3 hormone

1058

Estradiol

1068

Sertoli cell morphology

1068

Sperm counts

1068

Vimentin

1068

Vinculin

1068

Cell death

1080

Cryptorchidism

1080

Ischemia

1080

Testicular ischemia

1080

Azoospermia factor (AZF)

1088

Infertility

1088

Male infertility

1088

Y-microdeletion

1088

Azadirachta indica

1093

Epididymes

1093

Leaf extract

1093

Litter size

1093

Astonia macrophyll

1104

Motility inhibition

1104

b-Sitosterol

1104

Leydig cells

939

Leydig cells 993

Sperm

1001

Sperm 1016

Oxidative stress

963

Oxidative stress 1058

Spermatogenesis

993

Spermatogenesis

1032

Male contraception

1032

Male contraception 1042

Testis

1048

Testis 1058
Testis 1093

Spermatozoa

963

Spermatozoa 1023
Spermatozoa 1093
Spermatozoa 1104
Germ cells 1048

Germ cells

1080

 

NISCAIR Policy on Plagiarism

 

The system of formal communication in science through publication in primary journals is based on originality and quality of information being the only criteria for publication. However, there have been tendencies to misuse the system and vitiate the process of science communication for personal benefits. On of the ills afflicting science communication is plagiarism. Attempts at plagiarism may range from verbatim, copying of extensive material of other authors, misappropriating results/data of others with minor changes in language/presentation without giving credit to original source, to publish essentially the same information more than once.

As the premier publisher in India of primary scientific journals in various disciplines of science and technology, NISCAIR strongly reiterates its policy of discouraging plagiarism of all kinds. All efforts are made detect and frustrate attempts at plagiarism through editorial screening and rigorous peer review in respect of communications received for publication in NISCAIR publications. Cooperation of the scientific community is sought in our efforts to frustrate all attempts at plagiarism.

In case any attempt to plagiarize is brought to our attention accompanied with convincing evidence, following steps would be taken:

 

§      After consulting the respective Editorial Board Members, authors guilty of plagiarism will be debarred from publishing their papers in NISCAIR journals

§      Heads of the departments/institutes of the offending authors will be intimated of such incidences of plagiarism.

§      Such incidents of plagiarism will be publicized through the concerned NISCAIR journals in consultation with the respective Editorial Board Members.

 

—————————————

 

Editor’s Note

 

          The Indian Journal of Experimental Biology is being covered by some of the following leading abstracting and indexing journals/ current awareness services ‑‑

          BIOSIS

          Chemical Abstracts Service

          Excerpta Medica

          Informascience

          Refrativnyi Zhurnal

          Zoological Records

          NISCAIR website (http://www.niscair.res.in)

          PUBMED (http://www.ncbi..nlm.nih.gov)

          MEDLINE

—————————

Announcements

 

National Symposium on Biodiversity and Insect Pest Management

2 and 3 February, 2006, Loyola College, Chennai

 

With a view to explore the emerging trends in pest management practices through biotechnology and other novel techniques, the Entomology Research Institute, Loyola College, Chennai is organizing the national symposium. The topics to be discussed are (i) Insect resistant transgenic crops, (ii) Microbial and botanical pesticides, process and development, (iii) Natural enemies and new techniques for the production of natural enemies, (iv) Hybridization technique in the production of potential natural enemies, (v) Insect and animal vectors of diseases, and (vi) Biosafety concerns. For further details, please contact Dr S. Ignacimuthu, s.j., Director, Entomology Research Institute, Loyola College, Chennai 600 034, India. Telefax: 044-28174644, E-mail: eri_lc@hotmail.com

 

———————————————

 

National Conference on Aquatic Resources, Aquaculture and Aquashow

(CONFAQUA 2006)

15–17 February 2006, Palayamkottai, India

 

With a view to understand the development of technologies and to develop strategies for better technology transfer, the titled conference will include following topics: (i) Fish germplasm, management and conservation, (ii) Integrated coastal zone management, quality and safety of sea food, (iii) Socio economics and marketing and policies and regulations, (iv) Breeding, genetics and biotechnology, (v) Nutrition, farming systems and husbandry practices, (vi) Environment, quarantine and fish health management, and (vii) Aquaculture in practice. For details please contact: Dr. M.A. Haniffa, Convener, CONFAUQA 2006, Centre for Aquaculture Research and Extension (CARE), St. Xavier’s College (Autonomous), Palayamkottai 627 006, India. Phone/Fax: +91 (0462) 2560670; Mobile: +91 (0462) 94431 – 57415; E-mail: rihani2003@yahoo.co.in

 

————————————

 

 

From the Guest Editor’s desk………

 

The subject, male reproductive biology, is too vast and is not possible to cover the entire gamut of research being carried out in this field in one issue of the journal. Thus, the scope of the present special issue of the Indian Journal of Experimental Biology remains restricted, and it aims to provide present state of the art of the subject only in limited areas of male reproductive biology. The issue comprises nine review articles and seven research papers covering wide range of topics.

The review articles deal with the role of thyroid hormones in Leydig cells steroidogenesis, detrimental effects of oxidative stress on male fertility, hazardous effects of endocrine disruptors in males, role of FSH in regulation of spermatogenesis and Leydig cell function, proteins implicated in sperm capacitation, and evaluation of sperm function for assisted conception; further, how does human sperm nuclear DNA integrity affect embryo quality has also been discussed. The current status of research in male contraception is also covered. The topics of research papers include male germ cell apoptosis, experimental hyperthyroidism-induced oxidative stress and impairment of antioxidant defence system, effects of oestradiol on expression of cytoskeletal proteins during spermatogenesis, germ cell death and removal during initial stages of testicular ischemia and cryptorchidism, male infertility: Y-chromosome deletions and testicular aetiology in cases of azoo-/oligospermia, and effects of plant extracts on male reproductive organs, and sperm motility.

All the authors, who have contributed articles in this special issue, are acknowledged scientists/researchers in their respective fields. I wish to express my personal gratitude to all of them for their contribution and overwhelming support and encouragement. This special issue could not have been completed without their fullest co-operation. My thanks are also due to my research students, Dr. Raghav Mishra and Ms Akanksha Singh, for their help. I will be failing in my duty if I do not acknowledge my wife and children who enabled me to concentrate in editing of this special issue.

I am extremely grateful to the Director, National Institute of Science Communication And Information Resources (NISCAIR), CSIR, New Delhi, for providing me an opportunity to be a Guest Editor of this issue. My sincere thanks are also due to the entire team of the Indian Journal of Experimental Biology for extending full support in undertaking this herculean task. Words fail to express my gratitude to Dr M K Singhal for his constructive suggestions and efforts for fast follow-up action to bring out this issue in time.

Finally, I owe the greatest debt to my revered teacher, Professor C. J. Dominic, FNA, who introduced me to the field of male reproductive biology. His guidance helped me to undertake this challenging task and hope this special issue will be useful to those interested in the area of male reproductive biology.

 

Shio Kumar Singh

(Guest Editor)

Department of Zoology

Banaras Hindu University

Varanasi - 221 005, India

 

Phone: +91-542-2307149 Extn. 131

Fax: +91-542-2368174

Email: shioks@rediffmail.com

shio@bhu.ac.in

——————————

 

 

Indian Journal of Experimental Biology 

Vol. 43, November 2005, pp. 939-962

 

 

 

Review Articles

 

Leydig cells, thyroid hormones and steroidogenesis*

S M L Chamindrani Mendis-Handagama & H B Siril Ariyaratne

 

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin.releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 963-974

 

 

Mechanism, measurement, and prevention of oxidative stress
in male reproductive physiology

Ashok Agarwal & Sushil A Prabakaran

 

Numerous factors influence male fertility. Among these factors is oxidative stress (OS), which has elicited an enormous interest in researchers in recent period. Reactive oxygen species (ROS) are continuously produced by various metabolic and physiologic processes. OS occurs when the delicate balance between the production of ROS and the inherent antioxidant capacity of the organism is distorted. Spermatozoa are particularly sensitive to ROS as their plasma membrane contains polyunsaturated fatty acids (PUFA), which oxidizes easily. They also lack cytoplasm to generate a robust preventive and repair mechanism against ROS. The transition metal ions that are found in the body have a catalytic effect in the generation of ROS. Lifestyle behaviours such as smoking and alcohol use and environmental pollution further enhance the generation of ROS and thus, cause destructive effects on various cellular organelles like mitochondria, sperm DNA etc. This article analyzes the detrimental effects of OS on male fertility, measurement of OS and effective ways to decrease or eliminate them completely. We have also provided information on oxidative stress in other systems of the body, which may be applied to future research in the field of reproductive biology.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 975-992

 

 

A treatise on hazards of endocrine disruptors and tool to evaluate them

Partha Roy & Ben M J Pereira

 

Hormones mediate a major part of our essential physiological functions. Both endogenous and exogenous compounds and their metabolites are known to act through hormone receptors leading to regulation of endocrine function. The endogenous ligands that control reproductive functions are generally steroids such as 17β-estradiol, androgens, progesterone, pregnenolone and glucocorticoids. However, exogenous compounds that are structurally and functionally similar gain entry into animals including humans through the diet or by occupational exposures, causing endocrine disruption. In the recent decade, there is a lot of apprehension about the so-called “endocrine disruptors” which are wide spread in the environment, mainly due to unrestricted human activity. These compounds of anthropogenic or natural origin mimic the action of sex hormones and can interfere with the endocrine system. It has been hypothesized that environmental exposure to synthetic estrogenic chemicals and related endocrine active compounds may be responsible for malformations in the male reproductive tract, crytorchidism, hypospadias, decrease in sperm counts, decreased male reproductive capacity and even testicular cancers. The increasing concern in both public and scientific communities about these abnormalities have prompted the initiation of epidemiological studies to not only identify, but to also analyze the short and long term effects of endocrine disruptors. As a result, a number of assays have been developed and are undergoing validation aimed at high throughput screening of chemical agents that disrupt endocrine activity. This review consolidates the findings of epidemiological studies, particularly in relation to male reproductive disorders and brings to light the various types of in vitro and in vivo models that are available for tiered testing of suspected compounds.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 993-1000

 

 

FSH, the neglected sibling: Evidence for its role in regulation of spermatogenesis and Leydig cell function

V Sriraman & A J Rao

 

The role of follicle stimulating harmone(FSH) in male reproductive function remains a matter of debate although recent evidences strongly suggest a role despite the controversies that arose following the results obtained with FSH-β null mice and observations from human FSH receptor mutations. This review summarizes the recent developments of our understanding on the role of FSH in male reproduction. Specifically the results obtained with FSH-β and FORKO null mice are be discussed in light of our observations employing active and passive neutralization of endogenous FSH in rodents and primates along with other studies.On the basis of results obtained employing a variety of models it can be conclude unequivocally that FSH regulates Leydig cell function and is essential for normal quantitative spermatogenesis.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1001-1015

 

 

Proteins implicated in sperm capacitation

Kasturi Mitra & S Shivaji

 

Spermatozoa after being deposited in the female reproductive tract spend a considerable time in this foreign environment prior to fertilization of the oocyte. Chang and Austin independently observed1,2 that this time spent by the spermatozoa in the female tract is not consequential but a necessary event in the life cycle of the male gamete, and Austin2 first called this maturation period of spermatozoa as ‘Capacitation’. Ever since, attempts have been made to understand and unravel the molecular mechanism of capacitation. Based on the results obtained so far, it is clear that capacitation is guided by novel signal transduction pathways influencing varied aspects of spermatozoa. Capacitation could be, thus, defined as the cumulative molecular, cellular and physiological changes that occur in spermatozoa in the female reproductive tract to achieve the final competence to fertilize the oocyte. This review is structured so as to first understand the key features of capacitation and then to survey the players which bring about these changes during capacitation.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1016-1022

 

 

Does human sperm nuclear DNA integrity affect embryo quality?

 

Silpi Acharyya, Sagarika Kanjilal & Asok K Bhattacharyya

 

Abnormalities in the male genome are a clear potential reason for post fertilization failure. Male infertility may arise due to high levels of loosely packaged chromatin and damaged DNA. The achievement of a correct chromatin packaging level is essential for successful fertilization. The chromatin contained in the nuclei of mammalian spermatozoa is an extremely compact and stable structure. The reports on mammalian spermatozoa indicate that available volume is insufficient to contain sperm chromatin packed in nucleosome like structure and thus is organized in a special way. Different unique properties of sperm DNA like high degree of inertness and stability, absence of transcription, replacement of somatic histone by protamine etc have made the study of sperm chromatin more interesting. Increased levels of sperm nuclear DNA damage exist in infertile men with abnormal sperm parameters (i.e. concentration, motility and morphology), and various assay techniques have been developed to evaluate sperm chromatin maturity/DNA integrity. These assays are based on the facts that defects in chromatin structure have been shown to lead to increased DNA instability and sensitivity to denaturing stress. DNA integrity in the sperm is essential for the accurate and successful transmission of genetic information. Importance of sperm DNA has also become more obvious in the context of assisted reproductive techniques. While recent advances in assisted reproductive technologies have made possible and practical for many infertile men to become father, the risk of transmission of genetic mutation to the offspring, however, still remains. Further research is necessary to devise techniques for identification and selection of sperm with undamaged DNA for ICSI or to remove sperm with damaged DNA from the semen sample to improve the pregnancy outcome in ICSI.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1023-1031

 

 

Current trends in evaluation of sperm function: In vitro selection and manipulation of male gametes for assisted conception

Alex C Varghese, Barnali Sinha & Asok K Bhattacharyya

 

With increasing medical utilization of assisted reproductive technology (ART), scientists and clinicians have been able to study extensively multiple cell functions operating synchronously and flawlessly during the events preceding, before and after fertilization. Critical evaluation of the functional status of spermatozoa for in vitro techniques such as sperm-mucus interaction, acrosome reaction status, sperm-zona pellucida binding and penetration tests, hyaluronic acid binding assay, and computer assisted semen analysis etc. can direct a male partner of an infertile couple to more aggressive forms of treatments. In vitro selection of functionally competent sperm cells is a pre-requisite for successful outcome in in vitro fertilization or in intracytoplasmic sperm injection (ICSI). Direct injections of acrosome-intact spermatozoa into oocyte during ICSI bypassing the normal events of sperm oocyte interaction and fusion events have raised concerns with regard to fertilization abnormalities and genetic issues. The present communication briefly reviews the sperm function tests with emphasis on its correlation with fertility outcome, and the currently employed sperm selection and manipulation procedures which may have implications in assisted conception programs.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1032-1041

 

 

Male contraception: Expanding reproductive choice

M Rajalakshmi

 

The development of steroid-based oral contraceptives had revolutionized the availability of contraceptive choice for women. In order to expand the contraceptive options for couples by developing an acceptable, safe and effective male contraceptive, scientists have been experimenting with various steroidal/non-steroidal regimens to suppress testicular sperm production. The non-availability of a long-acting androgen was a limiting factor in the development of a male contraceptive regimen since all currently tested anti-spermatogenic agents also concurrently decrease circulating testosterone levels. A combination regimen of long-acting progestogen and androgen would have advantage over an androgen-alone modality since the dose of androgen required would be much smaller in the combination regimen, thereby decreasing the adverse effects of high steroid load. The progestogen in the combination regimen would act as the primary anti-spermatogenic agent. Currently, a number of combination regimens using progestogen or GnRH analogues combined with androgen are undergoing trials. The side effects of long-term use of androgens and progestogens have also undergone evaluation in primate models and the results of these studies need to be kept in view, while considering steroidal regimens for contraceptive use in men. Efforts are also being made to popularize non-scalpel vasectomy and to develop condoms of greater acceptability. The development of contraceptive vaccines for men, using sperm surface epitopes not expressed in female reproductive tract as source, still requires considerable research efforts.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1042-1047

 

 

Perspectives of contraceptive choices for men

 

N K Lohiya, B Manivannan, S S Bhande, S Panneerdoss & Shipra Garg

 

Apart from condoms and vasectomy, which have several limitations of their own, no other methods of contraception are available to men. Various chemical, hormonal, vas based and herbal contraceptives have been examined and few of them have reached the stage of clinical testing. Promising leads have been obtained from testosterone buciclate/undecanoate, alone or in combination with levonorgestrel butanoate or cyproterone acetate, RISUG, an injectable intravasal contraceptive and a few herbal products, particularly the seed products of Carica papaya. It is feasible that an ideal male contraceptive, that meets out all the essential criteria will be made available to the community in the near future.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1048-1057

 

 

Papers

 

Mouse model of male germ cell apoptosis in response to a lack
of hormonal stimulation

Amiya P Sinha Hikim, Yanira Vera, Rashid i Elhag, Yanhe Lue, Yu-Gui Cui, Vanisha Pope, Andrew Leung, Vince Atienza, Christina Wang & Ronald S Swerdloff

 

Received 5 August 2005

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1058-1067

 

 

Experimental hyperthyroidism-induced oxidative stress and impairment of antioxidant defence system in rat testis

Dipak K Sahoo, Anita Roy, Shravani Bhanja & G B N Chainy

 

Received 30 August 2005

Short-term hyperthyroidism, induced by daily administration of T3 (20mg/100g body weight) for one, three, and five days consecutively, modulates oxidative stress and antioxidant defence parameters in mitochondrial and postmitochondrial fractions of testis in adult rats. Alteration in antioxidant defences along with oxidative stress parameters in testis by thyroid hormone was found to be associated with a decline in the number of sperms and disturbances in histoarchitecture of seminiferous tubules in the testes; the results indicated that induced hyperthyroid state altered testicular physiology by influencing antioxidant defence system of testes.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1068-1079

 

 

Effect of estradiol on expression of cytoskeletal proteins during spermatogenesis in testis of sexually mature rats

J Choudhuri, M Aleem, V Padwal, P Das Gupta, R D’ Souza, S Pathak, N Balasinor, M K Gill-Sharma

 

Received 7 February 2005

Earlier studies had shown that long term treatment with estradiol arrests spermatogenesis in adult male rats, at a dose of 0.1 mg/kg/day. The present study was therefore undertaken to ascertain the causes underlying the reduction in sperm counts by administering estradiol for a short term, at the dose of 0.1 mg/kg/day. Estradiol valerate was injected at a dose of 0.1 mg/kg/day, for a period of 10 days to one group of adult male rats, which were administered saline for 12 days prior to estradiol injection, and sacrificed after 22 days. The control group was administered saline for 22 days. The sera were analyzed for testosterone and FSH levels. One testis of each male was immersion fixed for histology, and for immunohistochemistry of two testicular cytoskeletal proteins, vimentin and vinculin. The contralateral testes were used for analysis of vimentin and vinculin gene expression by reverse transcriptase polymerase chain reaction (RTPCR) and western blotting. Another group exposed to estradiol for 10 days was injected with bromodeoxyuridine (BrdU), at a dose of 100mg/kg/day, to ascertain the effect on germ cell proliferation, and sacrificed 12 days later, while estradiol treatment was continued till sacrifice. BrdU, at a dose of 100mg/kg/day was injected i.p. to a group of control rats treated with saline for 10 days, and sacrificed 12 days later. The testes from both groups were immersion fixed for immunohistochemical detection of BrdU. Histology of estradiol treated testis showed predominance of tubules with round spermatids with accumulation of lipid droplets in Sertoli cell cytoplasm and decreased cell height, whereas controls showed elongating spermatids. BrdU immunolocalization in the testis, irrespective of treatment, indicated its incorporation in deoxyribonucleic acid (DNA) suggesting that estradiol sustained germ cell proliferation. Both vimentin and vinculin could be immunolocalized to the testis. The testicular levels of vimentin and vinculin, quantified after western blotting, were unaffected. The testicular expression of vimentin and vinculin seen by RTPCR was also unaffected. The study suggested that estradiol induced reduction in sperm counts was not due to adverse effects on proliferation. The observed predominance of seminiferous tubules showing round spermatids, accumulation of lipid droplets as compared to controls suggested that reduction in elongated spermatids occurred through reduced spermiation and phagocytosis. The study also suggested that reduction in Sertoli cell height after short-term estradiol treatment was not due to reduced expression of vimentin and vinculin, which could be maintained by estradiol. However, reduction in Sertoli cell height could have been due to suppression of FSH and testosterone, implicated in the polymerization of vimentin and organization of vinculin, two cytoskeletal proteins involved in inter-Sertoli or Sertoli-germ cell junctions. The study suggested that disorganization of Sertoli cell cytoskeleton and reduction in the volume of Sertoli cells could be an important factor for reduced efficiency of spermatogenesis after exposure to estrogenic molecules.

 

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1080-1087

 

 

Germ cell death and their removal during initial stages of testicular ischemia and cryptorchidism: A comparative analysis

 

M M Misro, S P Chaki & Dinesh K Gautam

 

Received 7 February 2005

Germ cell death and their removal from the seminiferous epithelium are common in the affected testis in conditions of unilateral ischemia or cryptorchidism; the similarities and differences, however, have not been studied between these two conditions. The present study was designed to examine the severity of the effect on testicular germ cells during the initial stages of both ischemia and cryptorchidism, which have significant implications on the restoration of fertility following surgical repair. Complete absence of spermatids was observed following 12 hr of ischemia as compared to 7 days of cryptorchidism. Germ cell removal in either case was in the direction of lumen to basement membrane leaving only a single layer of cells by 24 hr of unilateral ischemia as compared to 15 days of cryptorchidism. Levels of intratesticular testosterone was found lower in cryptorchidism (7 days) but not in ischemia till 24 hrs. Giant cells frequently observed in cryptorchid testis were absent in the ischemic seminiferous epithelium. There was a gradual increase in the number of apoptotic and non-viable cells; the latter was more than 95% by 24 hr of ischemia. In contrast, approximately 85% testicular cells were nonviable till 15 days of cryptorchidism. The 1c peak representing the population of haploid cells was significantly reduced in cryptorchidism (7 days), while the peak was completely abolished by 24 hr of ischemia. Rise in the levels of oxidative stress in the affected testis was observed identically during the initial stages. These findings indicate that coupled with the rise in tissue oxidative stress, the number of apoptotic/nonviable germ cells was alarmingly high (>80%) by 15 days of cryptochidism or 24 hr of ischemia. Restoration of complete spermatogenesis following surgical repair may not be possible in such cases because of these acute adverse effects.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1088-1092

 

 

Male infertility: Y-chromosome deletion and testicular aetiology
in cases of azoo-/oligospermia

Kiran Singh & Rajiva Raman

 

Received 2 February 2005

The spermatogenesis locus azoospermia factor (AZF) in Yq11 has been delineated into three microdeletion intervals designated as AZFa, AZFb and AZFc. AZFc is the most frequently deleted region. We have studied 270 male infertile patients for various genetic disorders associated with infertile phenotype. In this study, we have presented results of our studies on Y-chromosome deletions, chromosomal abnormalities (Klinefelter syndrome) and histology of testis with the objective of seeing whether there were cases of gonosomic mosaicism and a causal correlation between the genetic disorder; and testicular aetiology could be drawn. In all the 13 cases of Y-chromosome microdeletion, AZFc region and DAZ gene were deleted, while no case of AZFa deletion was detected. This result was at variance with other reports from India, where a considerable fraction of cases showed deletion in AZFa region of the Y-chromosome. Both Y-deleted  and non-Y-deleted cases revealed heterogeneous testicular phenotype with comparable severity. This disparity among testicular phenotype in cases with known genetic aetiology and even in cases of unknown aetiology can be attributed to different genetic backgrounds and effect of modifiers. Since male infertility is a multifactorial disorder, the contributions of environmental and occupational insults may not be underestimated.

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1093-1103

 

 

Effect of aqueous leaf extract of Azadirachta indica on the reproductive organs in male mice

Raghav Kumar Mishra & Shio Kumar Singh

 

Received 19 September 2005

Effect of oral administration (50, 100, and 200 mg/kg body weight/day, for 28 days) of aqueous leaf extract of neem (Azadirachta indica) on the male reproductive organs of the Parkes (P) strain mice was investigated. The treatment had no effect on body weight and the reproductive organs weight. In treated mice, testes showed both normal and affected seminiferous tubules in the same sections; the affected seminiferous tubules showed intraepithelial vacuolation, loosening of germinal epithelium, marginal condensation of chromatin in round spermatids, occurrence of giant cells, mixing of germ cell types in stages of spermatogenesis and degenerated appearance of germ cells. In severe cases, the tubules were lined with Sertoli cells only, Sertoli cells and rare germ cells, or with Sertoli cells and several germ cells but without cellular association patterns. Also, the frequency of affected seminiferous tubules in testes of the extract-treated mice was significantly higher than the controls, though this remained unaffected in mice treated at 50 mg/kg body weight of the extract. Doses at 50 or 100 mg/kg body weight of neem leaf extract did not cause appreciable alterations in histological appearance of the epididymis, while a dose of 200 mg/kg body weight caused marked alterations both in histological appearance and the level of sialic acid in the duct. The treatment also had adverse effects on motility, morphology, and number of spermatozoa in the cauda epididymidis, level of fructose in the seminal vesicle, and on litter size. After 42 days of withdrawal of the treatment, the alterations induced in the reproductive organs recovered to control levels. Our results suggested that treatment with neem leaf extract caused reversible alterations in the male reproductive organs of P mice.

 

 

 

Indian Journal of Experimental Biology

Vol. 43, November 2005, pp. 1104-1109

 

 

Sperm motility inhibiting activity of a phytosterol from Alstonia macrophylla Wall ex A. DC. leaf extract: A tribal medicine

Debprasad Chattopadhyay, Sandhya R Dungdung, Kaushik Das, Sudipta Saha, Asit B Mandal &

Received 2 February 2005

The role of methanolic extract and n-butanol fraction of A. macrophylla leaves was investigated on the forward motility of goat spermatozoa. The methanol extract (600 ΅g/ml) and one n-butanol fraction (Fraction A; 100 ΅g/ml) showed marked inhibition of sperm forward motility, tested by microscopic and spectrophotometric methods. Approximately, 50-60% of the spermatozoa lost their motility when treated with 600 ΅g/ml of methanol extract or 100 mg/ml of Fraction A. The Fraction A at 400 mg/ml concentration showed complete inhibition of sperm forward motility at 0 min. The inhibitory activity increased with the increasing concentrations of the fraction. The motility inhibitory activity of the Fraction A was stable to heat treatment at 1000C for 2 min. The compound showed high inhibitory effect in the pH range 6.7-7.6. Fraction A also showed high efficacy for inhibiting human sperm motility, assessed by the microscopic method. The phytochemical analysis of methanolic extract of A. macrophylla leaves revealed the presence of sterols, triterpene, flavonoid, alkaloid, tannin and reducing sugar, while the Fraction A contains β-sitosterol, a common phytosterol. The results demonstrate that Fraction A (β-sitosterol) is a potent inhibitor of sperm motility and thus it has the potential to serve as a vaginal contraceptive.