Spermatozoa N
Spermatozoa N
Spermatozoa N
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)
16
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,
17
S.M.Wykes et al.
18
Sperm mRNA
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