Atypical structure of the nuclear membrane, distribution of nuclear pores and lamin B1 in spermatozoa of patients with complete and partial globozoospermia

Abstract

Globozoospermia is a form of male infertility characterized by spermatozoa with spherical heads lacking acrosomes. The aim of this study was to evaluate ultrastructural and molecular defects in different types of globozoospermia. Semen samples from 12 infertile patients (9 with complete globozoospermia and 3 with partial globozoospermia) and 10 normozoospermic men (control) were examined by transmission electron microscopy and immunocytochemistry with antibodies against lamin B1. The presence of lamin A and progerin was assessed by reverse transcription-PCR. Whole exome sequencing was performed in three patients. In semen samples with complete and partial globozoospermia, lamin B1 was observed at the periphery of sperm nuclei, whereas lamin A and progerin were absent. Nuclear envelope pores were found in spermatozoa from both patient groups, regardless of morphology and chromatin condensation, in contrast to the control group. Non-condensed chromatin was present in 51%-81% of cases of complete globozoospermia and in 36%-79% of cases of partial globozoospermia. Homozygous DPY19L2 and SPATA16 variants were identified in two patients with partial globozoospermia and one patient with complete globozoospermia. An atypical nuclear membrane with abnormal nuclear pore distribution and lamin B1 localization was observed in spermatozoa from patients with both complete and partial globozoospermia. The genetic defects in the DPY19L2 and SPATA16 genes detected in patients from both globozoospermic groups suggest a generalized disruption of nuclear structure in globozoospermia, highlighting the genetic and phenotypic similarities between complete and partial globozoospermia.

Keywords: DPY19L2; acrosome; globozoospermia; lamins; male infertility; nuclear pores; spermatozoa; teratozoospermia.

FIGURE 1

Transmission electron microscopy of spermatozoa in patients with “complete” (A, B, D, E) and partial (C, F) globozoospermia. In “complete” globozoospermia, spermatozoa have a spherical head with no acrosome. (A), a sperm with “immature” chromatin (ICh), (B) sperm with condensed chromatin (Ch). (D) [fragment of (A)] and (E) [fragment of (B)], the nuclear envelope (NE) consisting of the outer and inner nuclear membranes with nuclear pores. (C), spermatozoa with immature chromatin (Ich), elongated nucleus and a small acrosome not attached to the nucleus (A). (F) [fragment of (C)], the nuclear envelope is visible, consisting of outer and inner nuclear membranes and nuclear pores (NP). Scale: (A, B) 2 µm, (C)—5 µm, (D, E)—500 nm, (F)—1 µm.

FIGURE 2

Transmission electron microscopy of spermatozoa with normal head morphology in control group. (A), a sperm head with non-condensed (“immature”) chromatin (ICh); (B, C), sperm head with condensed chromatin (Ch). (D) [fragments of (A, B), respectively]. Chromatin is tightly bound to the nuclear envelope (NE), which is poorly visible and contains no nuclear pores. The nucleus is surrounded by a plasma membrane (PM), and the acrosome (A) is located between the plasma membrane (PM) and the nuclear envelope (NE). (F) (fragments of (C)), a section through the nuclear pocket (Po)–zone at the base of the sperm head, where the nuclear envelope (NE) borders the nuclear pocket zone (Po). Nuclear pores (NP) were detected only in the redundant nuclear envelope of the nuclear pocket (Po) zone. (C) centriole at the base of the sperm head. Scale: (A, E, F)–500 nm, (B) 2 µm, (C) – 5 µm, (D)–200 nm.

FIGURE 3

Immunofluorescence microscopy. Complete globozoospermia. Lamin B1 is detected along the entire periphery of the sperm nuclei [Lines (A, B)] and in the midpiece of the flagellum of some spermatozoa [line (B)].

FIGURE 4

Immunofluorescence microscopy. Partial globozoospermia. Lamin B1 is detected along the entire periphery of the sperm nucleus of elongating sperm.

FIGURE 5

Immunofluorescence microscopy. Normozoospermia. Lamin B1 is very weakly detected in the midpiece of the sperm flagellum and at the base of the sperm nucleus.

FIGURE 6

RT-PCR analysis of lamin A and progerin mRNA in spermatozoa.