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MHREP$0103

Molecular Human Reproduction vol.3 no.1 pp. 15–19, 1997

Haploid transcripts persist in mature human spermatozoa

Susan M.Wykes1, Daniel W.Visscher2 and Stephen A.Krawetz1,3


1Department of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, Wayne State University School
of Medicine, 275 E. Hancock, Detroit, Michigan, 48201, and 2Department of Pathology, Harper Hospital, Detroit, Michigan,
48201, USA
3To whom correspondence should be addressed
Mammalian spermiogenesis is marked by the morphological and functional differentiation of round haploid
spermatids into mature spermatozoa. A molecular restructuring of the chromatin accompanies this process
facilitated by the transition proteins and protamines which compact and condense the genetic material within
the developing spermatid. Previous studies from this laboratory have demonstrated that human protamines
PRM1, PRM2 and transition protein TNP2 transcripts are associated with round and elongating spermatids.
Extending this investigation, we examined the occurrence of these transcripts in mature spermatozoa by
in-situ hybridization analysis using [35S]-labelled cRNA probes. These results demonstrate that PRM1, PRM2
and TNP2 haploid-specific transcripts are present in mature spermatozoa. Quantitative analysis of the localized
signal also indicates that the PRM1, PRM2 and TNP2 transcripts persist at a similar ratio to that previously
described for these transcripts in human testes, i.e. PRM2.PRM1µTNP2. The persistence of these transcripts
in mature spermatozoa warrants further investigation.
Key words: haploid transcripts/in-situ hybridization/mRNA/protamines/spermatogenesis

Introduction PRM1, PRM2 and TNP2 transcripts are indeed still present in
Mammalian spermiogenesis is characterized by the morpho- mature spermatozoa. Quantitative analysis of the localized
logical differentiation of round spermatids into mature sperm- signal also suggests that PRM1, PRM2 and TNP2 transcripts
atozoa (Clermont, 1963). This process is mediated by the persist at levels consistent with the pattern of expression
transition proteins and the protamines which compact and previously described for these transcripts in human testes, i.e.
condense the genetic material into a species-specific shaped PRM2.PRM1µTNP2 (Wykes et al., 1995). The presence of
nucleus (Balhorn, 1989). The resulting mature spermatozoa transcripts in mature spermatozoa does not preclude an addi-
are essentially quiescent with respect to transcription (Bellvé tional function for their corresponding gene products within
et al., 1988). Despite the apparent transcriptionally inert state the sperm nucleus, subsequent to spermiogenesis. The basis
of the mature spermatozoa, the presence of RNA as well as for the persistence of transcripts in mature spermatozoa and
residual DNA polymerase activity have been described (Witkin their potential role during fertilization are discussed.
et al., 1975; Pessot et al., 1989). In human spermatozoa,
the presence of specific RNA transcripts including c-myc,
Materials and methods
leukocyte common antigen, protamine PRM2 and members of
the β1 integrin family, have also been suggested (Kumar et al., Molecular cloning
1993; Chiang et al., 1994; Miller et al., 1994; Rohwedder Subcloned fragments within the coding regions for the human
et al., 1996). Although the presence of RNA transcripts in protamines PRM1 and PRM2 and transition protein TNP2 transcripts
human spermatozoa is documented, there is little information of 443, 520 and 581 bp respectively were prepared as previously
available regarding their function within the mature sperm described by Nelson and Krawetz (1993). All fragments were initially
nucleus or during fertilization. subcloned into the Sma I site of pTZ18R (Amersham, Arlington
We have previously demonstrated that the human protamines Heights, IL, USA). The inserts were then excised from pTZ18R
using restriction enzymes, Eco R1 and Bam HI (Amersham) and
PRM1, PRM2 and transition protein TNP2 transcripts first
directionally ligated into the corresponding sites of pCR II (Invitrogen,
appear in association with round spermatids (Wykes et al.,
San Diego, CA, USA), which possess flanking SP6 and T7 bacterial
1995). This has confirmed their haploid-specific transcrip- phage promoters. In this manner, both sense and antisense transcripts
tion. While this study also established the presence of these could be generated from the same construct. A 269 bp fragment
transcripts in elongating spermatids it did not address their within the coding region for the human β-globin transcript was
subsequent fate. We have now extended these observations by synthesized using Perkin-Elmer PCR primer set PC04/GH20, then
in-situ hybridization analysis to establish the presence of all ligated into the PCR product cloning site of pCR II (Invitrogen). E.
transcripts of the PRM1→PRM2→TNP2 domain in mature coli, Top 10 cells (Invitrogen) were subsequently transformed with
spermatozoa. The results presented below demonstrate that the these constructs by electroporation as recommended by the manufac-
© European Society for Human Reproduction and Embryology 15
S.M.Wykes et al.

Figure 1. The effect of in-situ hybridization on sperm cell morphology. Bright-field photomicrographs at 3400 original magnification.
(A) Untreated sperm smear; (B) adjacent sperm smear hybridized with an [α-35S]-labelled sense probe. Spermatozoa subjected to the
hybridization process have slightly swollen heads and increased basophilia. All spermatozoa hybridized with [α-35S]-labelled control sense
probes showed a similar, sparse, non-specific distribution of silver grains.

turer (Invitrogen). Following isolation, all of these constructs were solution at a concentration of 253106/ml. Sperm smears were prepared
sequenced to confirm orientation. with 20 µl of this solution per slide using VectabondTM (Vector,
Burlingame, CA, USA) silane-coated slides and then allowed to air
Template preparation dry. The smears were then fixed in 10% neutral pH buffered formalin
Glycerol stocks of pCR II transformants were used to inoculate 30 ml for 5 min, rinsed in distilled sterile water for 5 min and then dried
of 23 YT media (2:4:1, yeast extract/tryptone/sodium chloride) and at 65°C for 1 h. Subsequent to drying, the slides were stored in a
grown at 37°C overnight. Template DNA was isolated using the desiccant-containing slide box at –80°C until use.
alkaline lysis method and then purified by differential salt precipitation
Pretreatment of slides
and phenol-chloroform extraction. Following ethanol precipitation,
residual RNA was removed by incubation with 3 U of RNase A Frozen sperm smears were allowed to thaw to room temperature,
(Amersham) at 37°C for 60 min. The PRM1, PRM2 and TNP2 then rinsed in RNase-free water. One sperm slide was incubated in a
templates were linearized by digestion with either Eco RI or Bam HI 20 µg/ml solution of RNase A in 10 mM Tris–HCl, pH 8.0 buffer,
(Amersham) and the β-globin template with either Bam HI or Not I containing 0.5 M NaCl plus 1 mM Na2EDTA at 37°C for 30 min,
(Amersham), generating their corresponding sense and antisense while the remaining slides were left in RNase-free water. All the
transcripts. Linearized pT3T719 and pTRI-β-actin (Ambion, Austin, slides were then digested with 1 µg/ml proteinase K (Gibco-BRL,
TX, USA) templates, both containing a 250 bp mouse β-actin coding Gaithersburg, MD, USA) at 37°C for 30 min, followed by acetylation
fragment, were used for the corresponding β-actin sense and antisense with 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine-HCl,
transcripts, respectively. Following enzymatic digestion, the template pH 8.0, at room temperature for 10 min. The pretreated slides were
DNA was purified by phenol-chloroform extraction and ethanol then rinsed in RNase-free water and dehydrated through a series of
precipitation. grades ethanol washes (50, 70, 95, 100%) and air-dried.
In-situ hybridization
Synthesis of cRNA probes
The in-situ protocol was adapted from that of Angerer and Angerer
cRNA probes were synthesized using the Maxiscript in-vitro (1992) as described (Wykes et al., 1995). In brief, the sperm smears
transcription system (Ambion) to a similar specific activity of were circumscribed with rubber cement and the cement allowed to
108 c.p.m./µg. In brief, after the addition of 1 µg of linearized solidify. Subsequently 200 µl of the hybridization solution composed
template and 80 µCi [α-35S]UTP (Amersham), the transcription of 20 mM sodium acetate, pH 5.2, buffer, containing 106 c.p.m. of
reaction mixture was incubated at 37°C for 60 min. Upon completion, the [α-35S]-labelled cRNA probe, 1 mg/ml tRNA, 0.1 M DTT, 50%
the DNA template was removed by treatment of the reaction mixture formamide, 300 mM NaCl, 50 mM Na2EDTA, 10% polyethylene
with 2 IU of RNase-free DNase I at 37°C for 30 min. The cRNA glycol (PEG) PEG-8000 plus 13 Denhardt’s solution (Amersham) was
probes were recovered by ethanol precipitation and then resuspended applied to each slide. The slides were then hybridized overnight in a
in 0.1 M dithiothreitol (DTT) to inhibit the oxidation of the [α-35S]- moisture-retentive chamber. The PRM1, PRM2 and TNP2 cRNA
labelled nucleotide probes. The theoretical Tm for each of the cRNA probes were hybridized at 56°C, while β-globin and β-actin cRNA
PRM1, PRM2 and TNP2 probes was calculated as 67.8, 68.7 and probes were hybridized at 42°C. Both sense and antisense probes were
68.9°C respectively. The theoretical Tm for the cRNA human β- simultaneously hybridized using separate slides. The RNase-treated
globin and mouse β-actin probes was calculated as 60.1 and 67.5°C slide was hybridized with the PRM2 antisense probe. A formalin-fixed
respectively. human bone marrow smear, serving as a positive control for β-globin,
was hybridized with the β-globin antisense probe. Following hybridiza-
Patient samples
tion, the non-specifically bound probe was removed through a series of
Liquified human semen samples, from a normal male donor, were progressively higher stringency washes. After the final wash, the slides
obtained from the in-vitro fertilization (IVF) clinic, Hutzel Hospital were rinsed in RNase-free water and dehydrated through a series of
(Detroit, Michigan). The specimens had an average concentration of graded ethanol washes, then allowed to air-dry.
153107 spermatozoa/ml, with motility .65% and containing ,1%
immature germ cells and lymphocytes. Sperm cells were washed in Autoradiographic detection
a solution composed of a 50 mM Tris–HCl (pH 7.5) buffer containing Subsequent to washing, the slides were coated with a thin film of a
2.5 mM MgCl2 plus 25 mM sucrose, then resuspended in the same 1:1 mixture of Kodak NBT-2 (Eastman Kodak, Newhaven, CT, USA)
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Sperm mRNA

Figure 2. In-situ localization of transcripts in human spermatozoa, (A) protamine PRM1, (B) protamine PRM2, (C) transition protein TNP2,
(D) β-actin and (E) β-globin. Bright-field photomicrographs at 3400 original magnification of human spermatozoa hybridized with the
corresponding [α-35S]-labelled antisense probes are shown. Silver grains indicating the presence of PRM1, PRM2, TNP2 and β-actin
transcripts were similarly distributed for each transcript over the entire head of the spermatozoon. In contrast, β-globin displayed a sparse,
non-specific distribution of silver grains similar to that of the sense controls, indicating the absence of β-globin transcripts in mature
spermatozoa.

emulsion in 0.3 M ammonium acetate prewarmed to 45°C, then was determined by measuring the grain density for sperm smears
allowed to dry at room temperature in darkness for 2–3 h. The slides hybridized with the corresponding [α-35S]UTP-labelled sense probes.
were then transferred to a light-tight slide box containing desiccant Ten sperm cells were measured per microscopic field and average
and exposed at 4°C for 2–3 days. Following autoradiographic develop- ROD values per field for each slide were then calculated. A final
ment, the sperm smears were counterstained using a haematoxylin mean of all 10 fields for each slide was determined and expressed as
and eosin histological stain. ROD 6 SE. Statistical significance was assessed using one-way
analysis of variance with Scheffé post-hoc tests.
Image analysis
Ten representative microscopic fields were selected at 332 magni-
fication using a Leitz Fluovert FU inverted microscope (Wetzlar, Results and discussion
Germany) and a Dage-MTI CCD 72 video camera (Michigan City,
IN, USA). The images were analysed using the Imaging Research, In-situ hybridization analysis was used to assess whether
MCID M4 Image Analysis Software Ver. 2.0 rev. 2.2. Silver grains human protamines PRM1, PRM2 and transition protein TNP2
indicative of specific RNA transcripts were quantified as a function transcripts were present in mature spermatozoa. Bright-field
of the relative optical density (ROD) for sperm smears hybridized with photomicrographs at 3400 magnification summarizing these
each respective [α-35S]UTP-labelled antisense probe. Background results are shown in Figures 1 and 2. As shown in Figure 1,
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S.M.Wykes et al.

results for each transcript were obtained by scanning 10


independent random fields and evaluating 100 individual
spermatozoa. They are summarized as a histogram shown in
Figure 3. The density of silver grains was above background
for each of the PRM1, PRM2, TNP2 and β-actin transcripts
(P ,0.0001). The density of silver grains for β-globin tran-
scripts and for the RNase-treated spermatozoa did not exceed
those of background. The level of PRM2 transcript was
significantly greater than those of PRM1, TNP2 and β-
actin transcripts (P ,0.0001), all of which were present
in approximately equal quantities. Interestingly, the relative
amount of each of the PRM1, PRM2 and TNP2 transcripts in
human spermatozoa is consistent with the pattern previously
described for these transcripts in human testes i.e.,
PRM2.PRM1µTNP2 (Wykes et al., 1995). It must be noted
that the testicular pattern of expression previously reported
for these transcripts was confined to round and elongating
spermatids and was not evaluated in testicular spermatozoa.
Paired analysis of PRM1, PRM2 and TNP2 transcripts in
Figure 3. The relative amounts of human protamines PRM1,
PRM2, transition protein TNP2, β-actin and β-globin transcripts in testicular and ejaculate spermatozoa are required before any
mature spermatozoa. Silver grains indicating the presence of direct comparison in the levels of expression can be determined.
specific RNA transcripts were quantified as a function of relative Using in-situ hybridization we have demonstrated that the
optical density (ROD) for spermatozoa hybridized with each human PRM1, PRM2 and TNP2 transcripts are present in
respective [α-35S]-labelled antisense probe. Background was mature spermatozoa. This is in accord with the reverse tran-
determined by measuring ROD for spermatozoa hybridized with
each corresponding [α-35S]-labelled sense probe. The ROD was scription (RT)–PCR data of others that showed that PRM2
measured for a total of 100 sperm cells over 10 microscopic fields transcripts are present in mature human spermatozoa (Miller
for each slide. The mean value of hybridization 6 SE was then et al., 1994) and that transcripts representing the various
determined for each transcript and is represented in the histogram members of the β1 integrin family are also present (Rohwedder
shown. et al., 1996). In addition to the above and the other previously
reported transcripts associated with human spermatozoa
spermatozoa subjected to the hybridization protocol have (Kumar et al., 1993; Chiang et al., 1994; Miller et al., 1994),
slightly swollen heads and increased basophilia. No other the presence of the PRM1, PRM2, TNP2 and β-actin transcripts
effects were obvious. The distribution of silver grains, now firmly establishes the presence and generality of RNA in
indicating the presence of human PRM1, PRM2, and TNP2 spermatozoa. The absence of β-globin transcripts and others
transcripts in mature spermatozoa, is shown in Figure 2. All (Rohwedder et al., 1996) implies that this is limited to a
displayed a similar distribution of silver grains deposited specific subset of genes. Although the relative amounts of
specifically over the entire head region of the spermatozoa. the PRM1, PRM2 and TNP2 transcripts in human testes
The patterns of hybridization for β-actin and β-globin tran- and mature spermatozoa are not directly comparable, the
scripts, which served as positive and negative controls respect- data suggest that, after spermiogenesis, these transcripts are
ively, are also shown in Figure 2. β-Actin transcripts, which sequestered in some manner while they await their fate. As
are constitutively expressed in most cells, were also distributed yet, there exists no information regarding the fate or potential
over the entire sperm head. In contrast, β-globin transcripts, role of these transcripts during fertilization and the formation
that are restricted to the erythroid cell lineage, displayed a of a viable male pronucleus.
non-specific sparse distribution of silver grains, similar to that
of the sense controls. These results, showing the presence of
Acknowledgements
PRM1, PRM2, TNP2 and β-actin transcripts and the absence
The Division of Reproductive Endocrinology and Infertility of the
of β-globin transcripts in mature spermatozoa, indicate that
Department of Obstetrics and Gynecology, Wayne State University
RNA in sperm arises from a specific subset of genes. The is gratefully acknowledged for providing human semen samples.
non-uniform distribution of silver grains over each cell is also
apparent. This may reflect the results of transcript sharing that
occurs among postmeiotic spermatids (Caldwell and Handel, References
1991) which could differentially load the individual sperm Angerer, L. and Angerer, R. (1992) In situ hybridization to cellular RNA with
radiolabelled RNA probes. In Wilkinson, D.G. (ed.), In Situ Hybridization.
with these transcripts, depending upon when the intercellular A Practical Approach. Oxford University Press, New York, pp. 16–32.
bridges were severed and then sealed. Balhorn, R. (1989) Mammalian protamines: structure and molecular
The relative amounts of human protamines PRM1, PRM2 interactions. In Aldolph, K.W. (ed.), Molecular Biology of Chromosome
Function. Springer, New York, pp. 366–395.
and transition protein TNP2 transcripts in mature spermatozoa
Bellvé, A.R., McKay, D.J., Renaux, B.S. and Dixon, G.H. (1988) Purification
were quantitated by measuring the intensity of the hybridization and characterization of mouse protamines P1 and P2. Amino acid sequence
signal as a function of relative optical density (ROD). The of P2. Biochemistry, 27, 2890–2987.

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Sperm mRNA

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Chiang, M.H., Main, E.K., Steuerwald, N. et al. (1994) Detection of human
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in human spermatozoa via reverse transcription polymerase chain reaction.
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Kumar, G., Patel, D., and Naz, R.K. (1993) c-Myc mRNA is present in human
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Miller, D., Tang, P., Skinner, C., and Lilford, R. (1994) Differential RNA
fingerprinting as a tool in the analysis of spermatozoal gene expression.
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Received on September 4, 1996; accepted on November 21, 1996

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