Download - A Study of mitosis, meiosis, histology, and scanning electron microscopic details of spermatogenesis in an infertile male with probable 46,XY/47,XXY germinal mosaicism

American Journal of Medical Genetics 10:119-131 (1981)

A Study of Mitosis, Meiosis, Histology, and Scanning Electron Microscopic Details of Spermatogenesis in an Infertile Male With Probable 46,XY/47,XXY Germinal Mosaicism Barathur R. Rajendra, Mingliang Lee, Louis Amorosa, and Leonard J. Sciorra

Division of Medical Genetics, Department of Pediatrics (B. R. R., M-I. L., L. J.S.), and Department of Medicine (L.A.), Rutgers Medical School, Piscata way, New Jersey

A 29-year-old infertile man with mild eunuchoid body proportions and unilateral gynecomastia with a low sperm count had normal chromosomes in lymphocytes and skin fibroblasts. Serum testosterone, urinary ketosteroid, and follicle-stimulating hormone levels were normal, but serum prolactin, luteinizing hormone, and estrogen levels were above normal. Testicular biopsy showed hypocellularity, a pacuity of maturing sperm, and increased lipid content in early spermatogonia. Cytogenetic analysis of the testicular material showed probable 46 ,XY /47 ,XXY mosaicism in spermatogonial cells. None of the cells in diakinesis and metaphase I showed the extra- X as a univalent, and 50% of the cells in diakinesis were hypodiploid with intact X Y bivalents. Further analysis of spermatogenesis revealed polymorphic dyads, triads, tetrads, and uncleaved meiotic products of first and second meiosis. Sex chromatin studies of testicular cells showed that 20% of the cells were X-chromatin positive. Scanning electron microscopic study of sperm revealed a wide range of polymorphisms, with some uncleaved meiotic products maturing into double-headed and double- tailed sperm or sperm with bulbous middle pieces or protrusions. A cytogenetic basis for the origin of these abnormalities is presented.

Key words: meiosis, spermatogenesis, scanning electron microscopy of sperm, sperm polymorphisms, infertility, germinal mosaicism, Klinefelter syndrome

INTRODUCTION Between 5% and 15% of all infertile men seen in infertility clinics have a chromo-

some abnormality [Koulischer and Schoysrnan, 19741 and asperrnia, azoospermia, oligo-

Received for publication November 27, 1979; revision accepted April 20, 1981.

Address reprint requests to Ming-liang Lee, MD, PhD, Division of Medical Genetics, Department of Pediatrics, CMDNJ-Rutgers Medical School, Piscataway, NJ 08854.

0148-7299/81/1002-0119 $04.00 0 1981 Alan R. Liss, Inc.

120 Rajendra et a1

spermia, asthenospermia, and/or teratospermia due t o abnormal spermatogenesis. One of the most common of these conditions is the Klinefelter syndrome. The manifestations of this syndrome may be reduced by XY mosaicism in somatic tissues. Some mosaic patients have been reported t o produce some motile spermatozoa and claim t o have fathered children [Foss and Lewis, 1971, Neuhauser and Opitz, 19751.

somatic cells and a probable 46,XY/47,XXY mosaicism in germ cells. We report a eunuchoid, infertile man with a 46,XY chromosome constitution in

REPORT OF PROPOSITUS

The propositus is a 29-year-old infertile man with normal physical and mental development and an unremarkable medical history. The patient has normal erections, ejaculations, and libido. He shaves daily. His sperm count was 1.2-2.0 million/cc; motility was reduced. The patient was treated with two injections of Depotestosterone (400 mg) 3 4 days apart; two months later sperm counts were not improved. The patient’s wife is normal and conceived after artificial insemination by donor.

Physically, the patient was healthy, well developed and of normal intelligence. Height was 183 cm and span was 187 cm; UBSlLBS ratio was 88/95 (0.92). (Father’s height is 183 cm and brother’s height is 185 cm.) Facial, axillary, and pubic hair distribution was normal; however, little hair was present on the chest or upper limbs. (Patient’s father and brother are very hairy.) The skin was soft, and the amount of subcutaneous fat was greater than normal. There was fullness of the left nipple, with palpable glandular tissue, but n o galactorrhea. External genitalia appeared normal. Both testes measured 19.7 cm3 (normal range 10.7-21.1 cm3) and were firm; the prostate was firm, smooth, and of normal size.

Endocrine studies showed the following results: serum testosterone level 540 ng/dl (normal 300-1,200); serum prolactin 41.6 ng/ml (7-1 8) ; urinary LH 35 IU/day (5-20); urinary FSH 1 5 IU/day (2-15); ketosteroids 14.4 mg/day (10-23); and total estrogens 136 mcg/day (4-25).

MATERIALS AND METHODS

Chromosome Analysis Routine chromosome analysis on cultured lymphocytes (banded and unbanded) and

buccal smear analysis from right and left cheeks was done twice, 11 months apart; 100 metaphases were counted each time, and five karyotypes were prepared from each sample. A skin biopsy was obtained from the left arm, and fibroblast cultures were initiated in routine manner; 1 0 0 fibroblast metaphases were counted, and five karyotypes were prepared and analyzed. Lymphocyte and fibroblast chromosomes were also studied by fluorescent methods [Uchida and Lin, 19741.

Two sections of a testicular biopsy were obtained from the right testis for histological, cytogenetic, and transmission electron microscopy (TEM) studies. For cytogenetic studies, fresh testicular tissue was minced with a fine scalpel and scissors, incubated in 0.6% sodium citrate solution at 22°C for 25 minutes, and processed for chromosome study by the method of Sasaki and Makino [1965] . Slides were also processed for G- and Q-banding. All spermatogonial, diakinesis, and metaphase spreads were counted, photographed, and karyotyped when possible.

Meiosis in 46,XY/47,XXY Germinal Mosaicism 121

Histological Study In addition to the above studies, very thin sections of the biopsy were processed

for X-chromatin staining (Feulgen, hematoxylin and eosin) by the methods of Plunkett and Barr [1956] and Myers 119591 ; 300 cells from the tubules were scored for X- chromatin bodies. Testicular biopsy slides from two age-matched normal men (29-3 1 years of age) were used as controls in all comparisons of histology and sex chromatin analysis.

Scanning Electron Microscopy (SEM) of Sperm After liquefaction at 37°C for two hours, the semen samples, obtained after three-

day abstensions from sex, were washed gently in three changes of Krebs-Ringer phosphate buffer (pH 7.2) at room temperature, with intermittent and mild centrifugation [Could, 19731. The samples were then fixed in 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) and mounted on coverslips treated with poly-L-lysine (PLL), which is a modifica- tion of the method used by Mazia et a1 [1975]. A 0.1% solution of PLL (P-1886, Sigma) in water was dispersed onto coverslips and allowed to stand for two hours in a moist chamber at room temperature. After thorough rinsing in running distilled water, the coverslips were mounted onto SEM stubs with conductive silver paint with the PLL side face- up. Small drops of the fixed sperm were placed on each of the stubs and left in a moist chamber for two hours. The samples were then dehydrated in gradual increases of ethyl alcohol and critical-point dried. The stubs were then coated with gold-palladium (80 A) and studied at magnifications of 1,000 -1 0,000 in an ETEC Autoscan SEM. One hundred sperm were studied per stub and classified for various abnormalities; 25 sperm were measured for head length and width. Representative pictures were taken. A control sample of semen from a fertile normal donor was processed similarly for SEM studies.

RESULTS AND DISCUSSION

Cytogenetic Studies Chromosome analysis of the patient’s cultured lymphocytes and skin fibroblasts

showed a 46,XY constitution, without mosaicism or structural abnormalities. No X- chromatin bodies were seen in buccal smear cells.

Cytogenetic analysis of the testicular biopsy is presented in Table I. A total of 55 spreads were anlayzed. The majority(35) were spermatogonia, and 20 cells were in

TABLE I. Cytogenetic Data From Testicular Biopsy

Phase Total 46,XY 47,XXY Hypodiploid Hyperdiploid a) Spermatogonia 35 8 23 2 2

Total 22 Bivalents + XY 21 Bivalents + XY Random loss

b) Diakinesis or metaphase-I 20 10 Cia 4b

aFour cells had a missing D bivalent; one cell showed a missing B group bivalent; and one cell showed a missing C group bivalent. bThese cells had intact XY bivalents, but the missing bivalents could not be identified.

122 Rajendra et a1

diakinesis or early metaphase I. Only two second-division metaphase spreads were identified; counts were unclear. Of the 35 spermatogonial spreads, 23 (66%) (Fig. la) had a count of 47 chromosomes, eight had a normal 46,XY count, and four were hypodiploid or hyperdiploid. The extra chromosome was a C-group chromosome. Attempts to G-band these spermatogonial metaphases failed to identify the extra C-group chromosome, but Q-banding identified the Y chromosome. In all 23 spreads with 47 chromosomes, the extra chromosome was of the same C-group size.

Fig. 1. (a) Spermatogonial spread with karyotype showing 47 chromosomes. (b) Spennatogonial spread with karyotype showing 46 chromosomes.


Of the 20 cells in diakinesis or metaphase I (Table Ib), ten were normal with 22 bivalents + XY (Fig. 2a), six showed 21 bivalents + XY constitution, and among these four cells showed missing D-group bivalents (Fig. 2b), one cell had a missing C-group bivalent, and one cell had a missing B-group bivalent. The four remaining cells showed random loss of autosomal bivalents with intact XY bivalents. None of these cells showed the extra C-group chromosome.

Fig. 2. (a) Cell in diakinesis with 22 I1 + XY. (b) Cell in diakinesis with 21 I1 + XY.

124 Rajendra et a1

Histological Studies of Testicular Biopsy

tubules. Other areas showed relatively normal-appearing tubules with reduced number of mature sperm and increased lipid content in spermatogonia. There was no interstitial fibrosis or apparent Leydig cell hyperplasia. Sex chromatin analysis was done on very thin sections of the biopsy using Feulgen or cresyl violet staining. Hematoxylin and eosin stained and toluidine stained slides served as controls. Of 300 Sertoli, Leydig, and germ cell interphase nuclei analyzed, 59 were X-chromatin positive (Fig. 3 a-d). X-chromatin studies of testicular biopsies from age-matched controls (from established normal fertile males) did not show X-chromatin bodies in Leydig, Sertoli, or germ cell interphase nuclei.

These X-chromatin studies, and the patient’s phenotype and endocrine abnormalities, suggest that the extra C chromosome seen in 66% of the spermatogonial spreads was an X chromosome and that the patient had 46,XY/47,XXY germinal mosaicism. The fact that we did not detect the extra X chromosome in any of the diakinesis or metaphase I spreads either as univalent, trivalent, or fragment(s) in- dicates that either 1) the X chromosome is lost, 2) such cells do not go through meiotic division, or 3) we failed to see such cells in our slides. However, Neuhauser and Opitz [1975] report that spermatogonia with 47,XXY can go through meiotic division. Their patient was a man with 46,XY/47,XXY somatic mosaicism with an atrophic testis and a 47,XXY son. A similar discrepancy was observed between spermatogonial counts and diakinesis or metaphase I counts in several 47,XYY men. The expected extra Y chromosome was never identified in diakinesis or metaphase I spreads by Melnyk et a1 [1969], Evans et a1 [1970], or Hulttn [1970]. In one case, Hulttn [ 19701 reported X Y / X W mosaicism in spermatogonial cells, which was judged on the basis of six G-sized chromosomes in some spermatogonial metaphases with 47 chromosomes. These results are very similar to our studies with a 46,XY/47,XXY mosaicism in germinal cells. Since the spermatogonial cells showed mosaicism, it is possible that this in itself causes meiotic instability (Table Ib).

In our patient’s testicular biopsy, we found a number of first-division and second-division chromatin and cell size polymorphisms, which included dyads, triads, and tetrads (Fig. 4 a-d). In plants, cell and gamete size polymorphisms arising from meiotic irregularities due to aneuploidy, pairing problems in prophase I , and disruption of meiotic spindles may result in sterility [Dover, 1972; Rajendra et al, 19781. Thus, the origin of cell size polymorphisms in our patient could be due to 47,XXY spermatogonial cells or aneuploid diakinesis. It is reasonable to assume that aneuploid germ cells give rise to gamete polymorphisms. These polymorphisms should be reflected at maturity as sperm size polymorphisms. Such findings were indeed apparent when the sperm were analyzed via SEM (see below).

studies. Furthermore, evidence of polymorphic differentiation was noted in the TEM studies (Fig. 5) and was confirmed in the SEM studies of sperm.

Light microsocpy showed areas of atrophy and hyalinization of the seminiferous

Material submitted for TEM analysis showed findings consistent with light microscopic

Light Microscopic and Scanning Electron Microscopic Studies of Sperm

results. Sperm motility varied between 35% and 50%. At least three different sperm sizes were observed.

Light microscopic studies of sperm on three different occasions yielded different


Fig. 3. (a) Two Leydig cells, one of which is sex chromatin positive (Feulgen staining; magnification, X 1,250). (b) A sex chromatin positive cell that is probably a secondary spermatocyte (Feulgen staining; magnification, X 1,250). (c) Sex chromatin positive and negative Sertoli cells. (Feulgen staining; magnification, X 1,250). (d) Cresyl violet-stained Leydig cells, one of which is sex chromatin positive (magnification, X 1,250).

Fig. 4. (a) A polymorphic dyad at the end of meiosis I. Note the area of noncleavage and distribution of chromatin. (b) A polymorphic triad at the end of meiosis 11. Note the point of noncleavage and micro-nuclei. (c) Half of a tetrad at the end of meiosis I1 showing noncleavage. (d) Half of a tetrad at the end of meiosis I1 showing differences in size of gametes (polymorphic).

126 Rajendra et a1

Fig. 5. A transmission electron microscopic picture of a section of testicular biopsy from patient showing abnormal sperm differentiation (magnification, X 8,000).

TABLE 11. Measurements of Sperm Heads (Width and Length) From the Patient and Normal Control Sperm*

Width Gum) Range Length Gum) Range

Control

Patient

~ ~~

2.4 k 0.023a (2.2-2.6) 3.4 f 0.025 (3.0-3.6)

2.7 k 0.098 (2.0- 3.2) 3.6 * 0.121 (2.3-4.5)

*Twenty-five sperm from controls and 25 sperm from the patient were randomly selected, photographed, and measured. aStandard error.

Various kinds of sperm cell size polymorphisms were detected in the SEM studies. Compared to control sperm from a normal fertile donor, our patient’s sperm showed higher head size variation (Table 11, Figs. 6 and 7). The length of the head also showed a wider variation than occurs in control sperm (Table 11).

In addition to these size polymorphisms, there were a number of morphological abnormalities. These included sperm with two tails (Fig. 7c), two heads and two tails (Fig. 6e), partially cleaved and large but mature sperm with well-differentiated tails (usually double tails) (Fig. 6c), and large sperm with bulbous middle pieces (as large as the head) and single tails (Fig. 6b). These abnormalities are classified in Table 111. Of special interest were the partially cleaved but fully mature sperm with two tails and the large sperm with bulbous middle pieces, which reflect some of the cell size polymorphisms observed in the testicular biopsy (Fig. 4). We think that such sperm originate in incompletely cleaved second meiotic divisions. Figure 6c shows a large sperm with a protrusion in the middle of the head and two well-differentiated tails representing a partially cleaved secondary spermatocyte. The two tails differentiate because genetic information is present in the uncleaved meiotic product; however, because of unequal distribution of chromatin or errors in gene expression, the two cells do not cleave completely, and


Fig. 6. (a) Morphology of sperm from normal fertile donor (magnification, X 10,000). (b) Sperm from patient showing bulbous middle piece and large head with single tail (magnification, X 10,000). (c) Sperm from patient showing protrusion in the middle of the large head (magnification, X 10,000). (d) Same sperm as in Figure 6c, showing the two tails at a lower magnification (magnification, X 4,000). (e) Sperm from patient with two uncleaved large heads and two tails (magnification, X 10,000). (f) Same sperm as in Figure 6e at a lower magnification, showing the origin of twin tails and size of sperm in relation to other sperm in the field. Note sperm (arrow) showing a protrusion in middle of head similar to one in Figure 6c (magnification, X 4,000).

128 Rajendra et al

Fig. 7 . (a) An apparently normal sperm from the patient. (b) A group of sperm from the patient showing sperm size polymorphisms. Note large sperm head at right top corner and small sperm at lower left corner. Also note double-tailed sperm (magnification, X 4,000). (c) Sperm from patient showing bulbous middle piece with two tails (magnification, X 10,000). (d) Sperm from patient showing bulbous middle piece with single tail (magnification, X 10,000). (e) A tissue piece of unknown origin in patient’s semen with lymphocytes and sperm attached to it (magnification, x 2,000).

maturation of the sperm occurs. This is also seen in Figure 6e, where the sperm has two large heads instead of protrusions, the point of non-cleavage is very clear, and the origin of the two tails is distinct. A diagrammatic representation of the origin of the various meiotic abnormalities observed in the present study and the origin of sperm cell size polymorphisms is presented in Figure 8.

and sperm from subfertile men show size variations in the modal range of 6.7-18.7 p m 3 . They state that one could speculate that oligospermia in such men reflects a fundamental defect that interferes with sperm maturation and that a correlation exists between lower sperm count and sperm size; ie, smaller sperm are associated with lower sperm count. Our studies d o not support this idea but indicate that low sperm count due t o aneuploidy in germ cells is associated with sperm cell size polymorphisms. We wholly agree with Koulischer and Schoysnian [ 19741 that cytogenetic studies on testicular biopsies from

Laufer et a1 [1978] reported that normal sperm has a modal volume of 15.2 pm3


TABLE 111. SEM Classification of Sperm From the Patient and From the Control Computed From a Total of 100 Soerm Each From the SEM Stubs

Pa tien t Con trola

“Normal-loo king”

Double heads with double tails

49 89

18b -

Double tails with single heads 13 2

Large heads with large bulbous middle pieces and single tails 19c 9d

“Age-matched, chromosomally normal, fertile donors.

bFive of these had partially obscured heads but welldefined tails; three of these were partially cleaved with protrusions in middle of the head.

%eludes extremely large middle piece bulb, large bulb, and medium bulb.

dThese sperm had megaloheads and did not look like sperm with bulbous middle pieces.

Fig. 8. A diagrammatic representation of spermatogenesis in the patient depicting the origin of various sperm polymorphisms and abnormalities.

130 Rajendra et al

infertile men with apparently normal somatic constitution could be very informational and may reveal a hidden chromosomal problem. Although the literature on the relationship between chromosome anomalies and sperm production is scanty, the study by Kjessler [ 19701 of a male carrying a pericentric inversion with azoospermia due to early prophase I degeneration is noteworthy. It is also worth mentioning two cases of infertility reported by HultCn et a1 [1970] showing azoospermia correlated with low chiasma counts, asymetri- cal bivalents, and univalents. In an earlier study done in 1964, Paulsen et a1 cultured germinal epithelial cells from the testes of a patient with 46,XY/47,XXY somatic mosaicism and found a similar mosaicism in the germinal epithelium. Gordon et a1 [1972] also observed similar mosaicism in cultured germinal epithelial cells from the testes in some infertile patients. While there have been several reports of testicular biopsy studies (non- cytogenetic) along with light microscopic analysis of semen [Foss et al, 1967; Foss and Lewis, 19711, ours is a report of a somatic 46,XY male whose infertility is due to polymorphisms and low sperm count as a result of probable 46,XY/47,XXY constitution of spermatogonial cells.

In light of the data obtained from our patient, we conclude that 1) chromosome analysis of testicular biopsy material could be beneficial in arriving at a diagnosis in some forms of infertility; 2) somatic 46,XY males whose infertility or subfertility is due to a 46,XY/47,XXY constitution in the spermatogonia alone represent a variant of the Kline- felter syndrome; and 3) the eunuchoid phenotype and the gynecomastia are probably due to the abnormal endocrine findings and the germinal 46,XY/47,XXY mosaicism.

ACKNOWLEDGMENTS

This study was funded by grants from the College of Medicine and Dentistry of New Jersey (CMDNJ 27-2625, MSRP 27-9870) and March of Dimes, Birth Defects Foundation (27-2587 and C-272/2-137). The authors wish to thank Dr. W. Burns, Lyons VA Hospital, New Jersey, for the preparation of TEM pictures, and Dr. L. S. Bates and Mr. John Krchma, Kansas State University, for letting the senior author use the SEM facility. We are grateful to Dr. A. C. Paulsen, USPHS Hospital, Seattle, Washington, for providing us with a number of slides from testicular biopsies (normal men), which served as controls in this study.

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Edited by John M. Opitz

Download - A Study of mitosis, meiosis, histology, and scanning electron microscopic details of spermatogenesis in an infertile male with probable 46,XY/47,XXY germinal mosaicism

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