MALE REPRODUCTIVE HEALTH AND ENVIRONMENTAL ENDOCRINE DISRUPTION

Project leader: Jorma Toppari, University of Turku, Departments of Paediatrics and Physiology, Kiinamyllynkatu 10, 20520 Turku, Finland, +358-2 333 7297, e-mail: Jorma.Toppari@utu.fi
 
 

PUBLICATIONS

TIIVISTELMÄ SUOMEKSI

Researchers:
University of Turku, Departments of Pediatrics and Physiology
Jorma Toppari, MD, PhD, docent, tel. +358-2-333 7297, e-mail: Jorma.Toppari@utu.fi
Marko Kaleva, MD, tel. +358-2-333 7297, e-mail: Marko.Kaleva@utu.fi
Helena Virtanen, MD, tel. +358-2-333 7297, email: Helena.Virtanen@utu.fi
Anne-Maarit Haavisto, MD, PhD, tel. +358-2-333 7297, e-mail: amhaavi@yahoo.com
Janne Suominen, MD, tel. +358-2-333 7297, e-mail:  Janne.Suominen@utu.fi
University of Turku, Department of Anatomy
Jyrki Suominen, MD, PhD, tel. +358-2-333 7228, e-mail: Jyrki.Suominen@utu.fi
Matti Vierula, PhD, tel. +358-2-333 7203, e-mail: Matti.Vierula@utu.fi
Johanna Järvi, lab. tech., tel. +358-2-333 7363
Antero Horte, MD, tel. +358-2-333 7203, e-mail: Antero.Horte@utu.fi

Consortium: Environmental health risks of dioxins
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 238316 / 862449
Person-months of work funded by SYTTY / Total person-months of work: 149 / 427

KEY WORDS: testis, sperm, cryptorchidism, environment, endocrine disruption
 

EXTENDED ABSTRACT

1 Introduction

Male reproductive health has deteriorated in many European areas during the recent decades, but the underlying reasons have remained elusive. Incidence of testicular cancer has increased rapidly, semen quality has declined, and prevalence of genitourinary malformations, such as hypospadias and maldescended testes, has increased. In Finland, the incidence of testicular cancer is small and semen quality has been better than in other European countries. Great regional differences and adverse temporal trends could be at least partially be explained by environmental influences. However, these factors are not known. Development and function of reproductive organs are controlled by hormones, and disruption of the endocrine system can theoretically cause all the problems of male reproductive health listed above. Animal experiments and some adverse effects of hormone therapy in humans have lent support to the hypothesis that environmental endocrine disrupters could be involved in the generation of reproductive problems. During recent years an enlarging group of  chemicals have been found to possess properties that could possibly disrupt normal endocrine signaling.

The aim of this research project has been to
Establish the current incidence of maldescended testes and hypospadias in Finland and to compare the Finnish figures to those from Denmark. At the same time, extensive data of the background factors and biological samples were to be collected in the prospective cohort study to find possible reasons for differences and to identify risk factors.

Compare the semen quality in different Finnish regions and in different European countries using well-harmonized methodology and external quality control. Again, extensive data of the background factors and biological samples were to be collected in the prospective cohort study.
Study the regulation of spermatogenesis and testicular steroidogenesis to find the putative target genes and functions that can be disrupted by environmental agents.

2 Methods

(1) The first objective has been studied in a prospective cohort study in which families have been recruited during the pregnancy. The cohort size prenatally recruited families was 1500 boys, and total cohort size was 5800 boys. The volunteering mothers were enrolled in the study when they returned a large questionnaire that gave information on their medical and reproductive history, work, diet, life style and other information that could give information on their exposure to different environmental factors. After birth the boys were examined during the first three days and again at three months. In a nested case-control study, the boys were examined for the third time at 18 months. Genitourinary malformations were recorded and the sizes of testes and penis were measured. For hormone and exposure analysis, the following biological samples were collected: mother’s serum during the first trimester of pregnancy, cord blood serum, placenta, breast milk, blood from the boy at three months. The hormone analyses included gonadotropins FSH, LH, polymorphic LH-variant, testosterone, sex hormone binding globulin, and inhibin-B. The exposure analyses will include dioxins, PCBs, polybrominated diphenyl ethers, halogenated hydrocarbons, alkylphenols, bisphenol A, selected pesticides and phthalate metabolites. Funding for exposure measurements has just started (EU) and it will be performed in Kuopio, Copenhagen and Munich.

(2) The second objective was investigated both in retrospective and in prospective studies. We first analyzed retrospectively semen quality in Finnish and Danish couples attempting to conceive. In prospective studies we have made semen analyses according to WHO guidelines and strictly controlled protocols in partners of pregnant women in Turku, Kuopio, Copenhagen, Edinburgh, and Paris. The study has been started also in USA and Japan. This study population has represented the fertile males who are supposed to have the best semen quality. To get information of general population, we have recruited military conscripts to semen analyses in Turku, Tartu, Kaunas, Copenhagen, and Oslo. In addition to semen analysis, the men were clinically examined, their reproductive hormone levels were measured (the same hormones as above in point 1), and their background data were collected by questionnaires. By harmonizing the methodology in multinational studies it has become possible to make international comparisons more reliably than previously.

(3) The third objective has required a large repertoire of cell and molecular biological techniques. We have used both in vivo and in vitro approaches. Testicular steroidogenesis has been studied in cultured tumor Leydig cells (MLTC). Gonadotropin responses have been studied by measuring cAMP and progesterone production of the cells. The effects of two common environmental chemicals, octylphenol and bisphenol A was analyzed. Regulation of spermatogenesis has been studied (1) in cultured rat and mouse seminiferous tubules, isolated from defined stages of the epithelial cycle by transillumination-assisted microdissection, and (2) in three in vivo animal models. These include ethylene dimethane sulfonate (EDS) –treated rats that are depleted of Leydig cells for days 2-15 after EDS administration, methoxy acetic acid (MAA) –treated rats, that are depleted of primary spermatocytes after treatment, and anti-c-kit-treated rats, in which the function of stem cell factor is inhibited by blocking the receptors. These models have given us good tools to analyze cellular interactions in the testis, regulation of the cell cycle and apoptosis. These are also key targets for environmental inluences that have been tested. Again, effects of octylphenol and bisphenol A have been studied. Components of the cell cycle and apoptotic machinery have been analyzed, as well as the most important paracrine factor, stem cell factor, its receptor c-kit, and androgen receptor interacting proteins. These proteins and their mRNAs have been mostly analyzed by immunohistochemistry, in situ hybridization, Northern and Western analyses, immunoprecipitation, PCR techniques. Apoptosis has been studied by in situ end labeling techniques and DNA electrophoresis. DNA synthesis has been assessed both by thymidine and bromodeoxy uridine (brdU) incorporation, and autoradiography plus brdU-immunohistochemistry.

3 Results and discussion

1. Developmental disorders of genital organs
We have completed clinical examinations the newborn boy cohort (1500 prenatally recruited boys and total cohort of 5800 boys) that was collected during 1997-99 (up to 18 months follow-up) and have started a parallel case-control study to get more subjects to study of developmental disorders of genital organs, particularly cryptorchidism. Relaxin-like factor (RLF, also called insulin-like hormone 3, Insl3) was shown to be important for testicular descent in experimental animals, and we analysed polymorphism of this gene in our patients. Polymorphism was found, but it was not associated to cryptorchidism (Koskimies et al., 2000). Now analyses of mutations in Insl3 receptor are on-going in collaboration with A. Hsueh in Stanford University. We obtained accurate data on the incidence of cryptorchidism and hypospadias in Finland, and the first reports were written (Toppari et al., 2001; Virtanen et al., 2001). The incidence rates were lower than in Denmark and several other Western European countries. Hormonal analysis comparing cryptorchid (maldescended testes) boys and controls revealed higher gonadotropin levels in abnormal boys, suggesting primary testicular failure that is compensated by gonadotropin stimulation in cryptorchid boys. We showed that early gonadotropin treatment may correct some of the disturbances in hypogonadotropic boys (Main et al. 2002). First measurements of dioxins in pooled breast milk samples from cases and controls did not show differences, but a comprehensive exposure analysis is now starting with EU funding.

2. Semen studies
We have verified large geographical variation in semen quality in Europe. First we demonstrated that semen quality of Finish men was better than that of Danes (Jensen et al., 2000). In  another multinational study semen quality of partners of pregnant women was analysed in Turku, Copenhagen, Edinburgh, and Paris. The study showed that Finnish men have better semen quality than others (Jorgensen et al., 2001). Time to pregnancy analyses (reflecting fertility) were performed in the same group, demonstrating correlation of semen quality to fertility (Slama et al., 2002) and differences in time to pregnancy times in Europe (Jensen et al. 2001). Differences in sperm morphology were also reported (Auger et al., 2001). Semen quality of military conscripts (age 18-19) in Turku, Tartu, Copenhagen, and Oslo showed surprisingly poor semen quality of the young men and east-west gradient in quality (better quality in Turku and Tartu than in Copenhagen and Oslo; Jorgensen et al, in press). In this material we have for the first time been able to find explanations for differences which appear to result from differences in fetal exposures (Jensen et al., submitted). We started follow-up studies to observe temporal trends. We also used archive material to assess the amount of abnormal sperm morphology during years 1980-94. There were no significant changes over time, but the younger age cohorts had more abnormal spermatozoa than the older birth cohorts (Horte et al., 2001).

3. Regulation of the development and function of the testis
We progressed fast in the studies of testicular cell death and cell cycle. We demonstrated the central role of stem cell factor (SCF) and Bcl2 family of proteins in the regulation of cell death in the testis (Hakovirta et al. 1999; Yan et al. 1999; Yan et al., 2000a-d). A comprehensive analysis of the role of retinoblastoma family of proteins in the cell cycle regulation of germ cells was also finished (Yan et al., 2001). Other important regulatory components included tetrapeptide N-acetyl-Ser-Asp-Lys-Pro and GATA-4 (Stephan et al., 2000; Ketola et al., 1999, 2000). Normal androgen signaling is crucial for the function of the testis. Androgen receptor interacting protein 3 was characterized (Moilanen et al. 1999), and several other interacting proteins have also been analyzed (Yan et al. Submitted). These regulatory components are important both as targets and mediators of endocrine disturbance. New molecular and cellular research methods were also developed (Zheng et al., 2000; Ventelä et al., 2000, 2001). Environmental chemicals, bisphenol A and octylphenol were shown to inhibit gonadotropin-stimulated steroidogenesis in Leydig cells in vitro (Nikula et al. 1999). These studies have now been extended to analysis of in vivo and in vitro effects of putative endocrine disrupters in fetal testis and ovarian follicles.

4 Conclusions

We have completed clinical examinations of a prospective birth cohort study that established reliable incidence rate of cryptorchidism for the first time in Finland. Identical study in Copenhagen enables us to make direct comparisons between Denmark and Finland, in which the incidence rates are very different (low in Finland, high in Denmark). Large background data, and several biological samples will give us information on the differences in hormonal levels, exposures, and potential risk factors between Finnish and Danish children and between cases and controls. Birth rate of hypospadias has also been established. We have also completed three sperm studies that have verified great regional variation in the semen quality. In all study groups (men of couples attempting to conceive, partners of pregnant women, and military conscripts), Finnish men had better semen quality than those from other participating Western European countries. Now we have also identified first causes for the differences that are related to exposures during pregnancy. These epidemiological studies form the baseline data on male reproductive health in Finland and will allow monitoring of any adverse trends. In experimental studies we have analyzed the regulation of cell death and proliferation in the testis and the key regulatory molecules that are involved. These determine the capacity of sperm production. We demonstrated that endocrine disrupters, such as octylphenols and bisphenol A can disturb Leydig cell steroidogenesis. This has important implications in the regulation of spermatogenesis that is dependent on high local androgen production. Androgen receptor interacting proteins and other testicular transcription factors may also be targets of endocrine disrupters, which is a studied in a new research project funded by the Finnish Academy (LIFE2000; Androgen signalling in the development and differentiation of the testis).
 
 

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