To better understand transcriptional regulation during human oogenesis and pre-implantation development, we defined stage-specific transcription, which revealed the cleavage stage as highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific, multi-copy retrogene, DUX4, encodes a transcription factor which activates hundreds of endogenous genes (e.g. ZSCAN4, ZFP352, KDM4E) and retroviral elements (MERVL/HERVL-family) that defines the cleavage-specific transcriptional programs in mouse and human. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells into two-cell embryo-like (‘2C-like’) cells, measured here by the reactivation of ‘2C’ genes and repeat elements, the loss of POU5F1 protein and chromocenters, and by the conversion of the chromatin landscape (assessed by ATAC-seq) to a state strongly resembling mouse two-cell embryos. Taken together, we propose mouse DUX and human DUX4 as major drivers of the cleavage/‘2C’ state.

During the elongating spermatid stage of spermiogenesis, human sperm chromatin undergoes a complex transition in which histones are extensively replaced by protamines in a carefully regulated transition including histone modifications and intermediate and temporary replacement of the histones by sperm-specific transition proteins. The replacement of most histones by protamines 1 and 2 facilitates a high order of chromatin packaging necessary for normal sperm function and may also be necessary for DNA silencing and imprinting changes within the sperm cell. Protamines 1 and 2 are usually expressed in nearly equal quantities, but elevated or diminished protamine 1/protamine 2 ratios are observed in some infertile men and is often associated with severe spermatogenesis defects. Human and animal studies demonstrate that expression of the protamine proteins is uniquely regulated by transcription/translation factors, including storage of the mRNA in ribonucleoprotein (RNP) particles composed of the mRNA, transcription factors and a kinesin molecule necessary for transport of the RNP to the cytoplasm and removal of transcriptional activators from the nucleus. Recent studies indicate that most patients with abnormal protamine protein levels have elevated levels of protamine transcript in the mature sperm cell, indicating a possible defect in transcription or translation. The regulation of protamine expression is unique and includes several possible mechanisms which may be responsible for dysregulation of protamine expression and concurrent broad spectrum defects in spermatogenesis. We suggest two hypotheses: (i) that abnormal protamine expression is indicative of a generalized defect in mRNA storage and/or translation which affects other mRNA transcripts or (ii) that protamines may act as a checkpoint of spermatogenesis.

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Background: Laboratory animals are commonly used for evaluating the physiological properties of the mammalian ovarian follicle and the enclosed oocyte. The use of different species to determine the morphological relationship between the follicle and oocyte has led to a recognizable pattern of follicular stages, but differences in follicle size, oocyte diameter and granulosa cell proliferation are not consistent across the different species. In an effort to better understand how these differences are expressed across multiple species, this investigation evaluates oocyte and follicle diameters and granulosa cell proliferation in the mouse, hamster, pig, and human.

Sperm protamine deficiency has been associated with human male infertility. However, most studies have adopted a global approach to assessing sperm protamine levels. Thus, it is not known whether sperm cells from individual human males possess variations in protamine protein content. The objectives of this study were to evaluate variations in protamine-1 (P1) and protamine-2 (P2) content between individual sperm cells of fertile and infertile men and to correlate DNA integrity and sperm cell viability with protamine levels in individual sperm cells. The semen samples of fertile and infertile men were evaluated globally for protamine protein content using nuclear protein extraction, gel electrophoresis, and densitometry analysis. Individual sperm cell P1 and P2 levels were assessed using immunofluorescence microscopy in conjunction with automated image analysis. The terminal transferase dUTP nick end labeling (TUNEL) assay was performed simultaneously with protamine immunostaining to assess the relationship between protamine levels and DNA integrity in individual spermatozoa. Additionally, the relationship between sperm cell viability and protamine levels was assessed via viability staining concomitant with protamine staining. The protamine fluorescence data demonstrate significant variations in protamine content within individual sperm cells of human males. Overall population-based measures of DNA integrity and sperm cell viability correlate significantly with population-based measurements of protamine levels. The data also demonstrate individual sperm cells displaying the lowest protamine levels display diminished viability and increased sperm cell susceptibility to DNA damage.

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.

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