The mammalian male reproductive system is comprised of the testis, encased in connective tissue Tunica albuginea and an intricate system of interconnected ducts. Both exocrine and endocrine in nature, the primary functions of the testis are to synthesize and secrete male sex hormones and generate sperms by the process of spermatogenesis. Sperm develops in the environment of the seminiferous tubules, which converges on the rete testis and leads out to a collective duct system called Epididymis [fig 1]. The Sertolli or Sustentacular cells lining the Epididymis sustain the proper environment required for spermatozoa development. One of the major roles of the Sertolli cells is to define two physiologically distinct compartments in the seminiferous tubule. Spermatogenesis is a continuous process by which primary male germ cells in the seminiferous tubules produce mature male gametes, the spermatozoa. It begins with diploid spermatogonial cells dividing by the process of mitosis to produce the primary spermatocytes. The primary spermatocytes generated by the process of spermatocytogenesis move into the tubule, where it’s DNA is duplicated. The primary spermatocytes then undergo meiosis I to produce haploid secondary spermatocytes. These haploid spermatocytes rapidly enter meiosis II to produce spermatids by the process of spermatidogenesis. Each spermatid condenses the DNA contained within it, develops a tail and an acrosome giving rise to an immotile spermatozoa [fig 2]. These are then transported to the epididymis where they become motile [1].
Phospho protein phosphatases, abbreviated as PPP, are enzymes that dephosphorylates certain phosphorylated proteins. Proteins are phosphorylated on nine amino acids, for example serine, threonine, tyrosine, cystine, etc. Of these, serine, threonine and tyrosine phosphorylation is predominant in eukaryotic cells and plays a role in key regulatory mechanisms. PPP1 belongs to protein serine/threonine phosphatases. Protein serine/thronine phosphatase is a big family of proteins found ubiquitously in eukaryotes. Protein phosphatase and protein kinase dynamically control the process of phosphorylation of structural and regulatory proteins in eukaryotes. Some of the physiological activities they are involved in include glycogen metabolism, calcium transport, muscle contraction and mitosis [2,11]. PPP 1 has four catalytic subunits PP1a, PP1ß, PP1¿1 and PP1¿2 [fig 3]. PP1¿1 and PP1¿2 are obtained by alternate splicing of the PP1¿ gene [fig 4]. The main structural difference between them lies in the C-terminus tail region [11]. PP1¿1 has seven exons while in PP1¿2, exon seven is internally spliced and translation of the mRNA adds an extra twenty one amino acid chain towards the 3’ UTR [13,14]. In terms of expression, PP1¿1 is expressed in Sertolli cells and PP1¿2 is expressed in developing germ cells. PP1¿2 is the most abundant isoform in the testis and an important regulator of sperm motility and function. PP1¿2 is also the only isoform detected in spermatozoa [5,12]. Previous research on mice has shown that PP1¿1 by itself is able to restore partial spermiogenesis but is unable to bring about motility and normal morphology, rendering those mice infertile. However, PP1¿2 by itself, in the absence of the ¿1isoform, is able to restore complete fertility [3].
Previous research, found PP1¿2 to be the most abundant isoform in testis and an important regulator of sperm motility and function. Targeted disruption of PP1¿ gene eliminated both spliced variants PP1¿1 and PP1¿2 and resulted in infertility in males due to impaired spermatogenesis and immotile sperms. However, females are unaffected, which implies that PP1¿ gene has a crucial role to play in the testis [4].
In order to determine which isoform of PP1¿ gene was the more important one, PP1¿2 was expressed as a transgene in mice lacking the PP1¿ gene, using a testis specific promoter called PGK2. PGK2, which stands for phospho gylcerate kinase 2, is a promoter which directs expression in testis [15]. Analysis of the results in terms of sperm count, morphology and motility, found that transgenic PP1¿2 males were fertile. This means that the absence of PP1¿2 is the main cause for impaired spermatogenesis [3].
Previous research has also shown that the amount of PP1 activity affects sperm motility. Too much activity renders them immotile but low activity levels initiate motility, as seen in caudal sperms [5]. The goal of the present work is to determine if over expressing PP1¿2 gene in a wild type (++) testis has any effect on spermatogenesis.
To go about doing that, wild type male mice were mated with heterozygous transgenic (+–/Tg+) females. This mating scheme was set up to produce mice with three copies, one transgenic and two endogenous copies of PP1¿2. Efforts have also been made to produce mice with four copies, two endogenous and two transgenic copies of PP1¿2. This is to see whether four copies further enhance the expression of PP1¿2 protein in the testis, that is, increase the production of sperms and their motility.