Mar 1, 1984 - Breeding mink for a fine dark fur has coselected male infertility, which may be manifest at the onset of breeding (primary infertility) or after one ...
0013-7227/84/1143-0922$2.00/0 Endocrinology Copyright© 1984 by The Endocrine Society
Vol. 114, No. 3 Printed in U.S.A.
The Dark Mink: A Model of Male Infertility* KENNETH S. K. TUNG, LEGRANDE E. ELLIS, GWEN V. CHILDS,t AND MARIA DUFAU Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131; Department of Biochemistry and Physiology, Utah State University, Logan, Utah 84322; Department of Anatomy, University of Texas Medical Branch, Galveston, Texas 77550; and Section of Molecular Endocrinology, Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, Bethesda, Maryland 20205
toimmune etiology. In contrast, fertile dark mink and fertile mink with the opaline and pastel fur have normal serum LH and testosterone levels; their testes are also normal. In mink with secondary infertility, the frequency and degree of orchitis and testiBularImmune complexes increased from March (peak sexual activity) to April (onset of testicular regression). Thus, testicular autoimmunity most likely develops during testicular regression. Antisperm antibodies also increased in frequency during testicular regression in the fertile dark mink and in dark mink with primary and secondary infertility. Thus, antisperm antibody per se is insufficient to induce autoimmune orchitis. It is concluded that the infertile mink is a useful model of human male infertility, involving both endocrinological and immunological mechanisms. (Endocrinology 114; 922,1984)
ABSTRACT. Breeding mink for a fine dark fur has coselected male infertility, which may be manifest at the onset of breeding (primary infertility) or after one or more fertile breeding seasons (secondary infertility). Mink with primary infertility have low LH and testosterone levels. However, they respond to exogenous GnRH with increases in LH production and in the number and size of LH and FSH positive gonadotropes in the anterior pituitary. Exogenous human CG also induces testosterone secretion. Thus, mink with primary infertility are probably defective in GnRH secretion, which is due either to abnormal hypothalamic function or its control mechanisms. Autoimmune orchitis with testicular immune complexes are frequent in mink with secondary infertility, suggesting an au-
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REEDING for mink with a dark fur has coselected male infertility. An earlier study of these animals in April 1980 has identified two clinical types: primary and secondary infertility (1). Mink with primary infertility were infertile at their first breeding season, whereas mink with secondary infertility were initially fertile for one or more breeding seasons. Mink with secondary infertility had high levels of antisperm antibodies, testicular immune complexes, orchitis, and aspermatogenesis. These findings indicate involvement of autoimmunity to sperm antigens as a pathogenetic mechanism. In contrast, evidence for immunological injury was not found in mink with primary infertility. The spontaneous infertile mink may be a model of male infertility. Infertile men may have abnormal hypothalamic-pituitary function in association with hypogonadism (reviewed in Ref. 2), which resemble the findings in the primary infertile mink. An association between antisperm antibodies and male infertility has also
been described (3,4). Moreover, a recent immunoelectron microscopic study on testicular biopsies from men with azospermia has disclosed frequently immunoglobulin deposits in tubular basement membranes, and this finding suggests the involvement of testicular immune complexes in human infertility (5, 6). The infertile mink is unique in having the features of testicular underdevelopment and testicular autoimmunity (1). In this study, we have investigated the hypothalamus-pituitary-testicular axis in the dark mink with primary infertility. In addition, we have studied infertile mink in March, at maximum sexual development. By comparing these results with the findings in April, when seasonal regression of the testis in normal mink is underway, we hope to gain further insight into the immunological processes leading to testicular autoimmunity in animals with secondary infertility. Materials and Methods Animals and experimental groups
Received April 7,1983. Address requests for reprints to: Dr. Kenneth S. K. Tung, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131. * This study was supported in part by NIH Grants HD-12247 and HD-15472 and a grant from the Mink Farmers Research Foundation. t Recipient of NIH Career Development Award HD-00362.
All animals used in this study were contributed by the Fur Breeder Agriculture Coop, at Midvale, Utah, and by Utah mink ranchers. Forty five mink (28 dark, 9 opaline, and 8 pastel) were used in the endocrinological study. These animals, born in 1981, were randomly chosen for this study in 1982. Serum 922
DARK MINK AND INFERTILITY levels of LH and testosterone were determined in February 1982. In early March, they were mated with receptive females to determine their reproductive performance. The semen of infertile mink recovered from the vagina either contained no sperm or a few poorly motile or immotile sperm. Thirteen of the fertile dark mink and 14 infertile dark mink were studied in mid-March for hormonal responses to exogenous GnRH and human CG (hCG). One hundred and thirty five dark mink were used in the immunopathological study. Of the 49 studied in March, 20 had primary infertility, 15 had secondary infertility, and 14 were fertile. Of 86 mink studied in April, 45 had primary infertility, 32 had secondary infertility, and 9 were fertile. All fertile mink in this study were aged 22 months. Data from animals studied in April have been described previously (1), but their reanalysis in the present study is necessary for comparison with findings of similar animals studied in March 1982. Hormonal assays To collect serum for LH and testosterone determinations, blood was placed on ice for exactly 60 min, and centrifuged at 15 min at 4 C. Serum was removed and immediately stored at —70 C until hormone determination. Testosterone was determined by RIA (7). Measurements of LH bioactivity in serum were performed by rat interstitial cell testosterone assay (8, 9). The recently described modifications that improved assay sensitivity by 5-fold (9) permitted accurate measurements of very low bioactive circulating LH levels in mink with primary infertility. Optional conditions for the bioassay of mink LH were provided essentially as previously described for measurements of bioactive serum LH in the rat. Briefly, the total incubation volume was 350 /A and contained about 2 x 106 interstitial cells per tube. LH-free serum was added to the standard curve and was also used to equalize the serum content of the assay. The sensitivity (detection limit) of the modified assay was 0.125 mg pure rat LH (Jutisz) or 2.5 ng RP-1. LH results are expressed in terms of RP-1 standard preparation provided by the Hormone Distribution Committee of NIADDK. Response to exogenous GnRH in vivo The animals were injected sc with GnRH (Beckman, Bioproducts, Palo Alto, CA) at a dose of 10 /ug/animal on Monday, Wednesday, and Friday for 2 weeks. Three days after the last injection, the animals were anesthetized with Ketamine, and each was injected im with 100 tig GnRH. Serum samples for LH and testosterone determination were collected from the tail artery immediately before and 35 min after the 100 ng GnRH injection. Response to exogenous hCG in vivo Animals were anesthetized with Ketamine and injected sc with 400 IU hCG (Pregnyl, Organon, West Orange, NJ) in 0.1 ml physiologic saline. Serum samples for testosterone determination were collected from the tail artery immediately before and 60 min after hCG injection.
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Immunocytochemistry of the pituitary Pituitaries were removed and immersed in 1% glutaraldehyde. They were cut into small blocks and then fixed for 1 h at room temperature. The blocks were then resuspended in several changes of buffer. This was followed by dehydration and embedding in Araldite 6005. The light and electron microscopic analyses were performed on semithin (1 /xm) and ultrathin sections stained immunocytochemically for LH/3 or FSH/3. The avidin-biotin peroxidase technique was applied as described (10,11). The same dilutions of antisera and antigens used in recent studies (10, 11), were applied to the mink pituitary sections, and they included: 1) antibovine LH/3, 1:10,000-1:20,000 + 10 /xg/ml FSH (Dr. J. G. Pierce, University of California, Los Angeles School of Medicine, Los Angeles, CA); and 2) antihuman FSH/3, 1:10,0001:20,000 + 10 Mg/ml LH (NIADDK, Dr. A. F. Parlow). Stained semithin sections were analyzed morphometrically as described recently (10) with a Zeiss microscope and a calibrated grid in the ocular. For each mink, 15-24 randomly selected fields were analyzed. The volume density (or volume fraction = Vv) of gonadotropic cells was determined by placing an ocular grid over the field and using the point counting method. The volume density (Vv) was calculated to be the number of points over a stained gonadotrope divided by the total number of points on the grid. It reflects changes in number and size of gonadotropes. The ocular grid was calibrated so that the volume density measurements could be converted to measurements of the percent of the grid area covered by individual gonadotropes (area fraction in /im). We calculated the percentage of LH cells and FSH cells in the population and measured the area fraction or average area of the grid covered by stained gonadotropes. The formulas are described in a previous paper (10). Detection ofantisperm antibody by indirect immunofluorescence (IF) Serum samples were obtained from anesthetized animals by cardiac puncture. Mink sperm were obtained by trimming cauda epididymides in NaCl (0.1 M, pH 7 at 20 C), washed once in saline, smeared on eight-hole teflon, slides (Roboz Surgical Inst., Washington, DC), and dried (12), The sperm smears were then fixed in absolute methanol for 15 min, dried, and stored at —70 C. Sera, diluted serially (2-fold) after a 1:10 dilution in PPS were incubated with the sperm smear for 30 min. After rinsing in PBS, the slides were incubated for 30 min with fluorescein isothiocyanate conjugated goat antiserum immunoglobulin G (IgG) to mink IgG. After rinsing in PBS, the slides were coverslipped with Tris buffered (pH 9.3) glycerol. The slides were studied with a Leitz fluorescence microscope with a Phloem illuminator. The highest serum dilution that gave an unequivocal positive fluorescence was the antibody titer. Studies on unthawed aliquots of sera of known antibody titer were included in each study to ensure reproducibility between studies. Direct IF study One testis or part of both testes and the cauda epididymis were snap-frozen in liquid nitrogen. Frozen sections of the
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testis were processed for direct IF as described previously (12). After fixation in ethanol-ether, the frozen sections were stained with fluorescein isothiocyanate-conjugated goat antisera IgG to mink IgG, mink IgM, IgA, and mink C3 [these reagents were generous gifts of Dr. David Porter, UCLA (Los Angeles, CA) and of Dr. John Coe, National Institute of Allergy and Infectious Diseases (Hamilton, MT)]. Histological study Three to five transverse sections through the testis, a section of the ductus efferentes, the caput and the cauda epididymides (fixed in Zenker's fixative) were processed for histology. Five micron sections were stained with periodic acid Schiff s hematoxylin. Approximately 500 cross-sections of seminiferous tubules per testis were examined. Statistical analysis Two-tail t tests, with unequal variance, were used to analyze the hormone levels; and the Student's t test was used for analysis of pituitary cells.
Results Hypothalamus-pituitary testicular function Hormone levels. LH and testosterone levels of 9 opaline, 8 pastel, and 28 dark mink, born in May 1981, were studied in February (1982) when testicular size and serum androgen levels of normal mink are maximum (13). The reproductive performance of these animals, studied 3 weeks later, revealed that all opaline and pastel mink were fertile. Of the dark mink, 13 of 28 (45%) were fertile, and their LH levels were comparable to that of the opaline and pastel mink (Fig. 1, left panel). In contrast, the LH levels of the 15 dark mink with primary infertility were significantly less than those of the fertile animals (P < 0.001). Similarly, testosterone levels in dark mink with primary infertility were significantly less than those in the opaline, pastel, and fertile dark mink (P < 0.001) (Fig. 2, left panel). These results indicate that primary infertility of the dark mink is associated with abnormal hypothalamic-pituitary-testicular function. We therefore determined the responsiveness of the pituitary and the testis of these animals to exogenous hormonal stimulation. Response to GnRH in vivo In order to prime the pituitary of infertile mink before acute stimulation with a large dose of GnRH, they were stimulated intermittently by small doses of GnRH for 2 weeks. As shown in Fig. 1, right panel, both fertile and infertile dark mink responded to 100 jug GnRH within 35 min. Whereas the preinjection levels of LH between the fertile and infertile animals were below the February level, they remained significantly different (P < 0.001).
OPAL PASTEL DARK IF)
COAT
TIME (mln)
FlG. 1. Serum LH levels in mink of different fur phases in February (left panel), at the time of peak sexual development, and the LH responses of fertile and primary infertile dark mink to GnRH in March (right panel). In February, the LH levels of the infertile (I) dark mink are significantly below that of the fertile (F) dark, opaline (opal), or ) fertile and infertile pastel mink. In March, the LH levels between ( ( ) dark mink are significantly different at time zero. In response to GnRH (100 fig), their LH levels were elevated to similar levels 30 min later.
OPAL PASTEL DARK
